Sequential injection system for the enzymatic determination of ethanol in wine

A sequential injection system was developed for the enzymatic determination of ethanol in wine. The spectrophotometric determination is based on the enzymatic reaction catalyzed by alcohol dehydrogenase in the presence of NAD+. The system was applied to the determination of ethanol in a range of 0.008-0.024% (v/v) with good repeatability; RSD(n=10) < 2.3%. The results obtained with the developed system showed good agreement with those obtained by using the reference method. The determination rate was 25 h(-1); 1 micromol of NAD+, 1.1 units of enzyme, and 50 microL of sample were consumed per determination; and the waste produced was 2.2 mL per assay.     

An automatic flow procedure for the determination of 3-hydroxybutyrate in animal serum and plasma

An automatic flow procedure based on the multicommutation concept, comprising three-way solenoid valves, for the spectrophotometric determination of 3-hydroxybutyrate in animal serum and plasma is proposed. The 3-hydroxybutyrate was enzymatically converted to acetoacetate with the reduction of NAD+ to NADH monitored at 340 nm. It was possible to carry out up to 600 determinations without a significant decrease in the analytical signal, with 5 mg of 3-hydroxybutyrate dehydrogenase immobilized on porous silica beads and packed in a column. The system enabled 60 determinations/h of 3-hydroxybutyrate in the range of 10-150 mg L(-1), with a consumption of 0.9 mg of NAD+ and 200 microL of sample per determination. A detection limit of 2 mg L(-1) for both animal serum and plasma and coefficients of variation of 1.4% and 1.2% (n = 17), respectively, were determined. Animal serum and plasma samples were analyzed without previous treatment, the results of which agreed with those obtained using the conventional method (UV kit, Sigma).     

Orthophosphate, phytate, and total phosphorus determination in cereals by flow injection analysis

A flow injection spectrophotometric procedure with enzymatic hydrolysis was developed for determination of orthophosphate, phytate and total phosphorus in cereal samples. Phosphorus species were extracted from cereals with 0.05 mol L(-1) potassium hydrogen phthalate buffer solution at pH 5.7. Orthophosphate was directly determined in the extracts by molybdenum blue spectrophotometric method. The phytate was hydrolyzed by the enzyme phytase coupled to a solid phase packed into an enzymatic reactor, and the resulting hydrolyzed orthophosphate was also determined by spectrophotometry at 650 nm. After optimization for phosphorus species extraction and enzymatic hydrolysis, a linear calibration graph was obtained up to 196 x 10(-6) mol L(-1) orthophosphate (P conc = -2.67 + 0.52x, r = 0.9998). Measurements are characterized by relative standard deviation of 1.6% for a standard of 72 x 10(-6) mol L(-1) orthophosphate and no baseline drift was observed during 4 h operation periods. It provides 72 measurements per hour, with 2.4 x 10(-)6) mol L(-1) and 7.9 x 10(-6) mol L(-1) as detection and quantification limits, respectively.     

Potentiometric flow injection determination of glycerol in distilled spirits.

A single-line flow injection system including a tubular periodate-selective electrode without inner reference solution is proposed for glycerol determination in distilled spirits, based on oxidation of this polyol by periodate. Interferences due to 5.0 mg L(-1) Cu, 5000 mg L(-1) sucrose, and 3000 mg L(-1) fructose plus glucose were investigated. The procedure is characterized by a linear response for 20-500 mg L(-1) glycerol (r > 0.9999, n = 7), a relative standard deviation of results of <0.03, and an analytical throughput of 30 determinations per hour. Accuracy was assessed by applying the procedure to distilled spirits of sugarcane and grape already analyzed by HPLC; in addition, recoveries within 96 and 120% were obtained.     

Phosphite determination in fertilizers after online sequential sample preparation in a flow injection system

A flow injection spectrophotometric system is proposed for phosphite determination in fertilizers by the molybdenum blue method after the processing of each sample two times on-line without and with an oxidizing step. The flow system was designed to add sulfuric acid or permanganate solutions alternately into the system by simply displacing the injector-commutator from one resting position to another, allowing the determination of phosphate and total phosphate, respectively. The concentration of phosphite is obtained then by difference between the two measurents. The influence of flow rates, sample volume, and dimension of flow line connecting the injector-commutator to the main analytical channel was evaluated. The proposed method was applied to phosphite determination in commercial liquid fertilizers. Results obtained with the proposed FIA system were not statistically different from those obtained by titrimetry at the 95% confidence level. In addition, recoveries within 94 and 100% of spiked fertilizers were found. The relative standard deviation (n = 12) related to the phosphite-converted-phosphate peak alone was 相似文献   

Development of an enhanced chemiluminescence ELISA for the rapid detection of acrylamide in food products

In this work, a polyclonal antibody for acrylamide (AA) was obtained by immunization of rabbits with N-acryloxysuccinimide (NAS) and keyhole limpet hemocyanin (KLH) conjugate. A direct enzyme-linked immunosorbent assay (ELISA) based on this antibody was developed with enhanced chemiluminescent (ECL) detection of AA in food samples. Assay conditions, such as concentrations of antibody and enzyme conjugate and competition time, were optimized. The effects of ionic strength and pH value were investigated. The optimized ECL-ELISA system allowed AA determination in a linear working range of 26.3-221.1 ng mL(-1) with an IC(50) value of 60.6 ng mL(-1) and a limit of detection of 18.6 ng mL(-1). Good recoveries with spiked food samples were obtained with a recovery range from 74.4 to 98.1%, and these results correlated well with those obtained using an HPLC method. This indicates that ECL-ELISA is applicable to the specific detection and routine monitoring of AA in food samples.     

Direct and selective flow-injection method for the simultaneous spectrophotometric determination of calcium and magnesium in red and white wines using online dilution based on "Zone Sampling".

The present work reports a selective and simple flow injection method for the direct and simultaneous determination of calcium and magnesium ions in red, rose, and white wines. Both ions react with methylthymol blue (MTB) at a strongly basic medium to form colored complexes that are monitored spectrophotometrically (lambda(max) = 610 nm). The simultaneous determination is achieved by online masking of magnesium by 8-hydroxyquinoline (8-HQ). Incorporating an online dilution mode based on the "zone sampling" technique in the FI system, the determination of both analytes was achieved without any pretreatment of the samples, in the range 0-350 mg L(-1) and 0-200 mg L(-1) for Ca(II) and Mg(II), respectively. The 3 sigma detection limits were quite satisfactory (2.1 and 1.8 mg L(-1) for Ca(II) and Mg(II) respectively), and the precision was 1.2% (at a mixture of 100.0 mg L(-1) Ca(II) + 100.0 mg L(-1) Mg(II), n = 12). A detailed study of interferences proved that the proposed method is highly selective. The application of the method to the direct analysis of red, rose, and white wines yielded excellent results compared with those obtained by using FAAS as a reference method (e(r) < 2.8%).     

Enzyme immunoassay for determination of gluten in foods: collaborative study

A collaborative study was performed in 15 laboratories to validate a monoclonal antibody-based enzyme immunoassay (EIA) for determination of gluten in foods. The study included 13 samples: maize starch, "gluten-free" baking mixes, wheat flours, cookies, cooked meats, and a soup. Gluten was present in these samples at either zero or 0.02 to 10% by weight, i.e., over almost 3 orders of magnitude. The mean assay values for the foods varied from 88 to 105% of the actual amounts. The assay was quantitative for cereal products and the soup with repeatability (RDS-r, relative standard deviation) and reproducibility (RSD-R) of 16-22% and 24-33%, respectively. The assay was semiquantitative for the processed meat products (RSD-r 14 and 26% and RSD-R 46 and 56%), probably because gluten was unevenly distributed in the small (1 g) samples that were analyzed. The ELISA method produced no false positive results, and false negatives obtained with tannin-containing foods could be avoided by use of a modified sample extractant. None of the collaborators reported problems in following the protocol. The method has been adopted official first action by AOAC for determination of wheat gluten in foods.     

Quantitative determination of L-arginine by enzymatic end-point analysis

An enzymatic end-point method for the quantitative determination of L-arginine was evaluated with samples of synthetic wine and natural grape juice. The enzymes arginase, urease, and glutamate dehydrogenase were used in this simple assay, similar to those described for many metabolites by Boehringer-Mannheim. In synthetic wine, recovery of L-arginine ranged between 98.3 and 104.4% and the precision as coefficient of variation was between 0.4 and 1.47% in the concentration range of the method, 0-100 mg/L L-arginine. The recovery of L-arginine in a grape juice with added L-arginine after clarification with polyvinylpolypyrrolidone ranged between 100 and 101.3%, and the coefficient of variation was 0.6%. The method has low material costs of approximately 0.43 U.S.$ per assay, and the time course of the reaction facilitates measurement of several samples concurrently. The results of this evaluation indicate that the enzymatic assay is a preferred method over colorimetric methods for the manual determination of L-arginine.     

Voltammetric determination of the antioxidant capacity in wine samples using a carbon nanotube modified electrode

A direct determination of gallic acid was achieved at a carbon paste electrode modified with carbon nanotubes under differential pulse voltammetry conditions. The values obtained for gallic acid were used to estimate the antioxidant properties of the wine sample based on gallic acid oxidation. The proposed method is based on the gallic acid oxidation process at a modified carbon paste electrode (MCPE) containing 30% (m/m) of carbon nanotubes monitored at 0.35 V versus Ag/AgCl (KCl 3 mol L(-1)). Using the optimized experimental conditions, the calibration curve for gallic acid was linear in the concentration range from 5.0 × 10(-7) to 1.5 × 10(-5) mol L(-1) with a detection limit of 3.0 × 10(-7) mol L(-1). The MCPE was successfully applied for the determination of the antioxidant capacity for red and white wine samples without interference of glucose and ascorbic acid, and the obtained results were compared with the standard spectrophotometric method.     

Enzyme immunoassay for hen egg white lysozyme used as a food additive

An indirect competitive enzyme immunoassay for hen egg white lysozyme (HEL) used as a food additive was investigated. Anti-HEL antibodies were obtained from B10A mouse ascites immunized by intraperitoneal injection of HEL. HEL samples to be assayed were extracted from foods with 1% gelatin in borate buffer. Goat anti-mouse IgG (H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 1-50 ng/mL, because in this range the binding inhibition curve of anti-HEL antibodies to HEL-coated plates by HEL was linear. Even after losing the lysozyme activity by heat treatment, HEL could be detected by indirect competitive enzyme immunoassay. Recoveries of HEL by this assay were greater than 85% for Japanese noodles and Japanese traditional-style confectioneries, 53-95% for Miso and cooked beans, and 30-85% for fried fish pastes. HEL contents of 55 commercial foods were determined; HEL was detected in 19 samples in the range 25-20,000 ng/g. HEL as a food additive was detected more frequently in plant-derived foods than in foods of animal origin.     

Solvent-free enzymatic preparation of feruloylated monoacylglycerols optimized by response surface methodology

The ability of immobilized lipase B from Candida antarctica (Novozym 435) to catalyze the direct esterification of glyceryl ferulate (FG) and oleic acid for feruloylated monoacylglycerols (FMAG) preparation in a solvent-free system was investigated. Enzyme screening and the effect of glycerol on the initial reaction rate of esterification were also investigated. Response surface methodology (RSM) was used to optimize the effects of the reaction temperature (55-65 degrees C), the enzyme load (8-14%; relative to the weight of total substrates), oleic acid/(FG + glycerol) (6:1-9:1; w/w), and the reaction time (1-2 h) on the conversion of FG and yield of FMAG. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of FG conversion and FMAG yield. The optimum preparation conditions were as follows: temperature, 60 degrees C; enzyme load, 8.2%; substrate ratio, 8.65:1 (oleic acid/(FG + glycerol), w/w); and reaction time, 1.8 h. Under these conditions, the conversion of FG and yield of FMAG are 96.7 +/- 1.0% and 87.6 +/- 1.2%, respectively.     

Development of an enzyme-linked immunosorbent assay based a monoclonal antibody for the detection of pyrethroids with phenoxybenzene multiresidue in river water

A sensitive and broad class selective direct competitive enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (McAb) has been described for the detection of pyrethroids with phenoxybenzene group. One monoclonal antibody, 2G(2)E(7), was obtained and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mice. The assay with the most selectivity for the family pyrethroids with phenoxybenzene group was optimized. The IC(50) values of the optimized immunoassay were 1.8 μg L(-1) for deltamethrin, 1.5 μg L(-1) for cypermethrin, 2.0 μg L(-1) for fluvalinate and fenvalerate, 2.2 μg L(-1) for phenothrin, 2.4 μg L(-1) for flucythrinate, 3.0 μg L(-1) for fenpropathrin, and 5.0 μg L(-1) for permethrin. River water samples fortified with pyrethroids were analyzed with the ELISA to evaluate the accuracy of the assay. The recoveries of pyrethroids in spiked water samples ranged from 74 to 108%. The results indicate that the ELISA developed can accurately simultaneously determine pyrethroids with phenoxybenzene group in water samples.     

Enzymatic determination of free glutamic acid in dried soups and in minced sausages: NMKL collaborative study.

An enzymatic method for the determination of free glutamic acid in meat products and dried soups was collaboratively studied in 11 laboratories. In the presence of the enzyme glutamate dehydrogenase, L-glutamic acid is oxidatively deaminated by nicotinamide adenine dinucleotide (NAD) to 2-oxoglutarate. In a reaction catalyzed by diaphorase, the NADH thus formed converts 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride to a formazan, which is measured in the visible range at 492 nm. Fourteen samples (7 samples of minced sausage and 7 samples of dried cauliflower soup) with glutamate contents varying between 0.4 and 16 g/kg were included in the study. Materials were distributed to participants as blind duplicates and as split level pairs. The mean relative standard deviation (RSDR) for reproducibility for the dried soup material containing glutamate between 7 and 16 g/kg was 4.6%. RSDR values for samples of minced sausage containing glutamate at lower levels (0.4-1.3 g/kg) were between 12 and 16%.     

Rapid time-resolved immunofluorometric assay for the measurement of creatine kinase in serum and whole blood samples

A rapid and simple immunochemical method was developed for the assessment of the creatine kinase (MM) isoenzyme [CK(MM)], a protein marker linked with animal welfare and meat quality. The one-step time-resolved immunofluorometric assay produced quantitative results from serum or whole blood samples in 20 min. The analytical limit of detection (mean + 2s) for the immunoassay was 17 ng/mL (n = 6), and the functional limit of detection for the analysis of porcine whole blood samples was 426 ng/mL (n = 24). The working range of the method was linear up to 50 micro g/mL, and the within-assay precision varied between 2.1 and 10.9%. The analysis of porcine serum samples showed that the results from the immunoassay method and colorimetric CK enzyme activity determination were highly correlated (r(2) = 0.965, n = 17, p < 0.001). The practicability of the assay was demonstrated by the analysis of 300 porcine whole blood samples in a slaughterhouse environment.     

Development of time-resolved chemiluminescence for the determination of antu in river water, wheat, barley, and oat grain samples

A novel chemiluminescence method for the determination of antu has been developed based on the reaction between potassium permanganate in acid medium with this rat-poison in the presence of formaldehyde as an emission enhancer. The main feature of the system used is that the recording of the whole chemiluminescence intensity-vs-time profiles can be obtained, using the stopped-flow technique in a continuous-flow system. This enables the use of three quantitative parameters adjustable via software settings, one of them a typically kinetic parameter, such as rate of the light-decay reaction, and the others conventional parameters, such as maximum emission intensity and total emission area, which are proportional to the analyte concentration. The optimum chemical conditions for the chemiluminescence emission were investigated. The effect of common emission enhancers, such as formic acid, formaldehyde, glutaraldehyde, acetaldehyde, quinine, fluorescein, rhodamine B, and rhodamine 6G, was studied. The parameters selected were sulfuric acid 4.0 mol L(-)(1), permanganate 0.1 mmol L(-)(1), and formaldehyde 1.0 mol L(-)(1). The calibration graphs obtained with each one of the measurement parameters were linear for the concentration range from 0.05 to 3.00 microg mL(-)(1). The detection limits ranged from 0.005 to 0.010 microg mL(-)(1), and RSD values (n = 10) of 0.99-1.79% at a 0.30 microg mL(-)(1) concentration level and 1.71-2.22% at a 1.0 microg mL(-)(1) concentration level were obtained. The present chemiluminescence procedures were applied to the determination of antu in different kinds of samples, such as river water, wheat, barley, and oat grain samples. Recovery values not significantly different from the spiked amount were found for these determinations.     

Monoclonal enzyme immunoassay for the analysis of carbaryl in fruits and vegetables without sample cleanup

The N-methylcarbamate pesticide carbaryl is one of the most important insecticides used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) for the determination of this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 101.0 +/- 26.9 ng/L and with a dynamic range between 31.6 and 364.0 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbaryl at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on both extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop and the fortification level, recoveries in the 59.0--120.0% range were obtained for purified samples and in the 70.0--137.7% range for crude extracts. The carbaryl immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbaryl at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was excellent (y = 1.04x + 0.71, r(2) = 0.992, n = 33), with HPLC being more precise than ELISA (mean coefficients of variation of 5.2 and 12.0%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.28x - 0.59, r(2) = 0.987, n = 33) while maintaining similar precision. Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbaryl in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases sample throughput.     

卵清蛋白基因表达放射免疫分析法

应用竞争结合原理建立了卵清蛋白基因表达的放射免疫分析法。采用氯胺T法标记卵清蛋白,~(125)Ⅰ-OA比活度达32～43μCi·μg~(-1),冻干后在常温下保存,有效期2～3个月;以(鸡)卵清蛋白作免疫原制备出的抗血清特异性强,与其他生物大分子无交叉反应,亲和常数为3.8×10~9L/M,50％结合率为1:1.6×10~5(最终稀释度);用双抗-PEG作分离剂沉淀免疫复合物,其方法灵敏度为0.17ng,可检范围为0.25～16ng,批内、批间变异系数分别为2.99％和8.96％。该方法能有效地直接检测类卵清蛋白含量,可作为一种特异、灵敏、简便、可靠的分析技术,应用于卵清蛋白基因表达生物基因工程研究。     

Determination of 2,4,6-trichloroanisole and 2,4,6-tribromoanisole in wine using microextraction in packed syringe and gas chromatography-mass spectrometry

A selective and fast method for the quantitative determination of 2,4,6-trichloroanisole (TCA) and 2,4,6-tribromoanisole (TBA) in wine was developed. Microextraction in packed syringe (MEPS) was optimized for the extraction and preconcentration of the analytes using extremely small volume samples (0.1-1 mL). For GC-EI-MS, the limit of detection (LOD) for red and white wine was in the range 0.17-0.49 microg L(-1) for TCA and TBA. In addition to GC-EI-MS both GC-NCI-MS and GC-HRMS were used to further improve both selectivity and sensitivity. The lowest LODs were achieved using GC-HRMS in the EI mode. In red and white wine samples the LODs were between 0.22-0.75 ng L(-1) for TCA and TBA. The reproducibility and linearity for the GC-HRMS method was good, with RSD-values of 4-10% for spiked red wine samples at 1 ng L(-1) and linearity with R (2) > 0.962 over a concentration range of 1 to 100 ng L(-1).     

Application of the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation assay to a flow injection system for the evaluation of antioxidant activity of some pure compounds and beverages

The 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(*)(+)) assay was adapted to a flow injection (FI) system to obtain a sensitive and rapid technique for the monitoring of antioxidant activity of pure compounds and complex matrixes, such as beverages and food extracts. The FI system includes a HPLC pump that flows the mobile phase (a solution of ABTS(*)(+) in ethanol) through a 20 microL loop injector, a single bead string reactor filled with acid-washed silanized beads, a delay coil and a photodiode array UV-visible detector. The technique was very sensitive, with limits of detection and of quantification of 4.14 and 9.29 micromol of Trolox/L, respectively, and demonstrated high repeatability and reproducibility. The proposed technique was then applied to the evaluation of the antioxidant activity of some pure compounds, demonstrating good agreement with published data obtained by the original spectrophotometric ABTS(*)(+) assay. Finally, the total antioxidant activity of 10 beverages was determined by both the proposed and the original method. The values ranged from 0.09 mmol L(-)(1) for cola to 49.24 mmol L(-)(1) for espresso coffee and did not result significantly different from those obtained by the original spectrophotometric ABTS(*)(+) assay (Student's paired t-test: t = 1.4074, p = 0.1929). In conclusion, the proposed FI technique seems suitable for the direct, rapid and reliable monitoring of total antioxidant activity of pure compounds and beverages and, due to the ability to operate in continuous, it allows the analysis of about 30 samples h(-)(1) making the assay particularly suitable for large screening of total antioxidant activity in food samples.