Development of a perfusion ion-exchange chromatography method for the separation of soybean proteins and its application to cultivar characterization

A perfusion ion-exchange chromatography method has been designed, for the first time, for the separation of soybean proteins and its application to the characterization of soybean cultivars. For that purpose, the gradient, the mobile phase composition (buffer concentration, buffer pH, and elution salt), and the temperature were optimized. The method consisted of a two-step gradient (0% B for 2 min and from 0 to 50% B in 10 min) being mobile phase A a 2 0mM borate buffer (pH 9) and mobile phase B a 20 mM borate buffer (pH 9) containing 1M sodium chloride. The procedure used for the preparation of sample solutions was significantly simpler than that proposed by other authors and basically consisted of dissolving in water. This method enabled the separation of soybean proteins from a soybean protein isolate in 11 peaks in about 9 min. The method was used to separate soybean proteins in different commercial soybeans. In general, the 11 peaks yielded by the soybean protein isolate were also observed in the chromatograms of all soybeans. However, the area percentages of every peak in every soybean enabled the differentiation between soybeans. Moreover, the method was also used to separate soybean proteins in the proteic fractions obtained from every soybean. Multivariate methods were used for patterns recognition and the classification of samples.     

Development of a capillary electrophoresis method for the determination of soybean proteins in soybean-rice gluten-free dietary products

CE has been applied for the first time to the simultaneous separation of soybean and rice proteins. Treated and untreated capillaries with different effective lengths as well as separation media at different pHs were tested. For that purpose, samples and standard solutions were prepared in 25:75 ACN-water media containing 0.3% v/v acetic acid. The use of an untreated capillary of 50 cm effective length together with an 80 mM borate buffer (pH 8.5) modified with 20% v/v ACN and UV detection at 254 nm were the conditions working the best. These conditions enabled the determination of soybean proteins in gluten-free dietary commercial products elaborated with soybean protein and/or soybean flour and rice flour using the standard additions calibration method. The method was linear up to 26 mg/mL of soybean proteins, the precision (expressed as RSD) was always better than 6%, and recoveries obtained for soybean proteins when spiking commercial products were very close to 100%.     

Determination of trace levels of dinitrophenolic compounds by microporous membrane liquid-liquid extraction in environmental water samples

A fast and simple hollow fibre-based microporous membrane liquid-liquid extraction (MMLLE) method is proposed for the determination of trace levels of dinitrophenolic compounds in water samples. The optimization step was performed using a three-variables Doehlert matrix design, involving the fibre length, the quantity of trioctylphosphine oxide (TOPO) in the acceptor phase and the extraction time. Using the established experimental conditions, some other parameters such as stirring speed, salt content, humic acids and different organic solvents as the acceptor phase were studied. Validation of the method included calibration experiments, linearity studies and determination of method LOD (MLD). The RSD was around 11% in all the experiments on different days at different concentrations. Separation and detection of four dinitrophenols were performed in 10 min with an RP-LC and a C(8 )column ACN-citric buffer gradient elution and diode array detection.     

Detection and quantitation of additions of soybean proteins in cured-meat products by perfusion reversed-phase high-performance liquid chromatography

Perfusion liquid chromatography has been applied in this work to the determination of soybean proteins in commercially available cured meat products, enabling the detection of additions of soybean proteins in cured meat products to which the addition of these vegetable proteins is forbidden and the quantitation of soybean proteins in cured meat products to which the addition of these proteins is allowed up to a certain limit. The analytical methodology is based on a sample treatment (fat extraction and soybean protein solubilization) prior to chromatographic analysis. Fat extraction with acetone and soybean protein solubilization with a buffer solution at basic pH (pH 10 or 9) were necessary to obtain selective and sensitive conditions. Use of water-acetonitrile-trifluoroacetic acid or water-tetrahydrofuran-trifluoroacetic acid linear binary gradients at a flow rate of 3 mL/min, a temperature of 50 degrees C, and UV detection at 280 nm enabled chromatographic analysis of soybean proteins in cured meat products in less than 3 min.     

Ultrasound-Assisted Extraction Combined with HPLC-UV for Fast Determination of Sulfamethazine and Its N4-Acetyl Metabolite in Plasma and Phosphate Buffer

A simple and cost effective HPLC-UV method for fast determination of sulfamethazine (SMZ) and its N₄-acetyl metabolite, N₄-acetyl sulfamethazine (AcSMZ), in plasma and phosphate buffer was developed and validated. The sample treatment was performed by ultrasound-assisted extraction with 1 mL ethyl acetate twice, each time 5 min. Moreover, there was no need for centrifugation and further clean-up, which shortened the measurement time. The total analysis time was less than 30 min. The method proved to be a selective and accurate tool for detection of SMZ and AcSMZ in plasma and phosphate buffer with satisfactory recoveries (85.43–99.29%) and little matrix effects (0.86–0.99). The proposed approach was also applied to the evaluation of pharmacokinetic and drug-metabolite protein binding interaction studies. The results show that SMZ and AcSMZ are non-concentration-dependent, and the metabolite does not bind competitively with the drug to the plasma protein.     

Use of dispersive liquid-liquid microextraction for the determination of carbamates in juice samples by sweeping-micellar electrokinetic chromatography

Dispersive liquid?Cliquid microextraction (DLLME) has been proposed for the extraction and preconcentration of 12 carbamate pesticides in juice samples, followed by their determination by micellar electrokinetic chromatography with diode-array detection. To improve sensitivity, an on-capillary sample concentration method based on sweeping has been developed. Also, separations were performed in an extended light path fused-silica capillary; the separation buffer consisted of 100?mM borate and 50?mM SDS (pH?9.0) with 5% acetonitrile. Samples were introduced by hydrodynamic injection, dissolved in the separation buffer, but free of micelles. Several parameters of the DLLME procedure (such as type and volume of extraction and dispersive solvents, pH, salt addition, and extraction time) were optimized. Recoveries obtained for fortified juice samples (banana, pineapple, and tomato) at three different concentration levels, ranged from 78% to 105%, with relative standard deviations lower than 9%. The limits of detection ranged from 1 to 7???g l^?1. Moreover, the method is fast, simple, and environmentally friendly.     

Development of a Simple,Underivatized Method for Rapid Determination of Free Amino Acids in Honey Using Dilute-and-Shoot Strategy and Liquid Chromatography-Tandem Mass Spectrometry

A simple, fast and reliable analytical method was developed for 20 free amino acids (FAAs) determination in honey samples through a dilute-and-shoot strategy and hydrophilic interaction liquid chromatography tandem mass spectrometry. Compared with previous reports, direct dilution by water has significantly reduced the matrix effect and facilitated full extraction of FAAs. Further, a 5 min determination method was established with an acetonitrile–water mobile phase system with 0.1% formic acid addition. The established method was validated and demonstrated several advantages including short detection time, wide linear range over 3–4 orders of magnitude, high sensitivity down to 0.1 ng/mL and negligible matrix effect. Twenty FAAs were determined in 10 honey samples from different botanical origins by this method, and 19 FAAs were found. This general applicable method was also promising for fast determination of FAAs in other practical samples.     

Development of a perfusion reversed-phase HPLC method for the separation of soybean and cereal (wheat, corn, and rice) proteins in binary mixtures. Application to the detection of soybean proteins in commercial bakery products

A perfusion reversed-phase HPLC method enabling the simultaneous separation of soybean and cereal (wheat, corn, and rice) proteins in commercial bakery products has been proposed for the first time. The method utilises an acetonitrile-water gradient containing an ion-pairing agent. Different ion-pairing agents were tried, 0.3% (v/v) acetic acid being observed to enable the separation of soybean from wheat, rice, and corn proteins while with 0.1% (v/v) trifluoroacetic acid only the separation of soybean and corn proteins was possible. Optimisation of the solubilisation conditions for proteins was achieved by testing different acetonitrile concentrations for the simultaneous extraction of soybean and cereal proteins: best recoveries were found with 25% (v/v) acetonitrile + 0.3% (v/v) acetic acid and with 40% (v/v) acetonitrile + 0.1% (v/v) trifluoroacetic acid. Chromatographic conditions such as gradient, temperature, and wavelength detection were also optimised. The method enabled the separation of soybean and cereal proteins in binary mixtures (soybean and wheat, soybean and corn, or soybean and rice proteins) in less than 5 minutes in a total analysis time of 20 min.     

On-line extraction and determination of carbofuran in raw milk by direct HPLC injection on an ISRP column

Summary A method has been developed for extraction and determination of carbofuran in milk. The method involved direct injection of raw milk on to a human serum albumin dimethyloctyl-silica gel (HSA-C₈) column and the use of 80:20 (v/v) 0.01 M phosphate buffer pH 5.5-acetonitrile as mobile phase. UV spectrophotometric detection was performed at 220 nm. Identification was based on retention time. Quantification was performed by automatic peak-area determination and was calibrated by use of an external standard.     

Analysis of bovine whey proteins in soybean dairy-like products by capillary electrophoresis

The simultaneous separation of bovine whey proteins [alpha-lactalbumin and beta-lactoglobulin (A+B)] and soybean proteins was performed, for the first time, by capillary electrophoresis. Different experimental conditions were tested. The most suitable consisted of 0.050 M phosphate buffer (pH 8) with 1 M urea and 1.2 mg/ml methylhydroxyethylcellulose, UV detection at 280 nm, 15 kV applied voltage, and 30 degrees C temperature. Quantitation of bovine whey proteins in a commercial powdered soybean milk manufactured by adding bovine whey to its formulation was performed using the calibration method of the external standard. Direct injection of a solution of the powdered soybean milk only enabled quantitation of alpha-lactalbumin in the commercial sample. Detection of beta-lactoglobulin (A+B) required acid precipitation of the solution of the sample in order to concentrate bovine whey proteins in the supernatant prior to the analysis of this protein in the whey obtained. Since alpha-lactalbumin could also be quantitated from the injection of the whey, the simultaneous determination of alpha-lactalbumin and beta-lactoglobulin (A+B) was possible upon acid precipitation of the powdered soybean milk solution. Detection limits obtained were 14 microg/g sol. for alpha-lactalbumin and 52 microg/g sol. for beta-lactoglobulin (A+B) which represent protein concentrations about 60 microg/100 g sample for alpha-lactalbumin and 100 microg/100 g sample for beta-lactoglobulin (A+B).     

Preliminary studies on selenium-containing proteins in Brassica juncea by size exclusion chromatography and fast protein liquid chromatography coupled to ICP-MS

An approach for screening and resolving selenium-containing plant proteins was developed based on the combination of sample preparation and multi-dimensional liquid chromatography coupled to ICP-MS. Different protein extraction protocols were investigated. A 24 h dodecylsulfate-mediated protein extraction in a sonication bath followed by acetone precipitation was found to be optimal. The use of different protein precipitate solubilizing agents (sodium dodecyl sulfate media and Tris-HCl buffer) demonstrates possible fractionation of the selenium-containing proteins. Selenium-containing protein screening and fractionation were carried out by means of SEC-ICP-MS. High molecular weight selenium containing proteins were solubilized with a sodium dodecyl sulfate-containing buffer, whereas the low molecular weight compounds were released into a Tris-HCl buffer. Size exclusion chromatography-fast protein liquid chromatography coupled to ICP-MS allowed separation and detection of several selenium-containing proteins in Se-supplemented wild type Brassica juncea plant, a fast growing selenium accumulator.     

Determination of berberine and strychnine in medicinal plants and herbal preparations by pressurized liquid extraction with capillary zone electrophoresis.

A simple and rapid method for the determination of berberine and strychnine in medicinal plants and herbal preparations for regulatory purposes using a home-made pressurized liquid extraction (PLE) system with capillary zone electrophoresis (CZE) using ultraviolet detection at 254 nm was developed. The effects of pH, concentration of buffer, and organic modifiers in the electrophoretic separation were investigated. The buffer used for CZE contained 50 mM ammonium acetate, pH 3.1. The effect of temperature on the extraction efficiency of strychnine in medicinal plants by PLE was demonstrated. Comparable or higher extraction efficiency was achieved with PLE for strychnine in medicinal plants and berberine in herbal preparations compared to soxhlet extraction. The effect of matrix interference in medicinal plants and herbal preparations containing a number of medicinal plants samples using CZE was investigated by standard additional experiments. The reproducibility of the method using PLE with CZE was found to vary between 2.4 and 10.7% (n = 5/6) for different types of samples on different days.     

Determination of percentage of RoundUp Ready soya in soya flour using real-time polymerase chain reaction: interlaboratory study

An interlaboratory study was conducted to evaluate a method for determination of the percentage of RoundUp Ready (RR) soya in soya flour using Taqman technology. The method included DNA extraction from the test portion with cetyltrimethylammonium bromide buffer followed by chloroform extraction and Wizard resin cleanup steps. The DNA was then assayed with primer and probe sets specific for lectin as the endogenous control and the RR insert as the target. The percentage of RR soya in the soya fraction of the sample was calculated by using a matrix-matched standard curve. Ten samples of split-level blind duplicates were sent to 22 laboratories in 12 countries worldwide. Test portions contained 0, 0.5, 0.7,1.6, 2, and 3.9% (w/w) RR soya prepared gravimetrically from commercially available RR standard reference materials. Based on the results for test materials, the relative standard deviation for repeatability (RSDr) for the method ranged from 9.3 to 19.3% and, for reproducibility (RSDR), ranged from 20.3 to 33.7%.     

Determination of pharmaceutical and personal care products in wastewater by capillary electrophoresis with UV detection

Capillary electrophoresis (CE) offers a fast and cost-effective alternative analytical technique to LC-MS/MS for separation and quantitation of many PPCP compounds in wastewater. In this study, we have developed a method that can simultaneously analyze eight different PPCP compounds in untreated wastewater (ibuprofen, triclosan, carbamazepine, caffeine, acetaminophen, sulfamethoxazole, trimethoprim, and lincomycin), using capillary electrophoresis with UV detection (CE-UV). The method detection limit (MDL) ranged from 1.6 to 68.7 ppb through solid phase extraction. The standard limit of quantification (LOQ) ranged from 0.63 to 7.72 ppm. Factors affecting separation and quantification of PPCPs, such as pH, electrophoretic potential, buffer strength, buffer type, and additives, were investigated and optimized. Water samples from two different wastewater treatment plants were collected and analyzed. The results obtained were comparable with those of LC-MS/MS. The technique developed in this study provides a low cost, simple, fast, and relatively sensitive method for determination of various PPCPs in wastewater samples for PPCP screening.     

Characterization and differentiation of diverse transgenic and nontransgenic soybean varieties from CE protein profiles

Nowadays, soybeans are commercialized in a wide variety of colors and tones. Moreover, some pigmented seeds are being commercialized as soybeans while, on other occasions, these seeds are labeled as mung beans, azuki beans or soybean frijoles generating confusion on their identity. In this work, CE has been applied for the first time for the characterization and differentiation of different pigmented beans commercialized as soybeans. Other seeds commercialized as azuki, mung green soybeans or soybean frijoles were also analyzed. Borate buffer (at pH 8.5) containing 20% v/v ACN was used as the separation media and solution containing ACN/water (75:25 v/v) with 0.3% v/v acetic acid was used to solubilize the proteins from the samples. A 50 cm bare fused-silica capillary was employed for obtaining adequate separations in about 12 min. The CE protein pattern observed for yellow soybeans was different from that corresponding to green and red soybeans. The seeds commercialized as black soybean presented electropherograms identical or similar to those yielded by the yellow seeds with the exception of the sample labeled as black soybeans frijoles that presented a totally different pattern. In addition, CE protein profiles obtained for azuki and mung green soybeans were very similar to those corresponding to red soybeans and green soybeans, respectively. Finally, the CE method was also applied to differentiate transgenic and nontransgenic soybean varieties. Discriminant analysis, using several protein peak areas as variable, was used to successfully classify these samples.     

Development of an ultra-high performance liquid chromatography analytical methodology for the profiling of olive (Olea europaea L.) pulp proteins

Ultra-high performance liquid chromatography (UHPLC) constitutes an interesting proposal to speed protein separations but it is almost not explored. In this work UHPLC is proposed, for the first time, to separate olive pulp proteins. An important difficulty in the analysis of proteins is related to their extraction. The difficulty in the extraction of proteins from the olive pulp is derived from its high content in lipids and phenolic compounds. Eight different methods for the extraction of pulp proteins were designed and evaluated. The optimized extraction procedure consisted of a cleaning step to remove interfering compounds, followed by the extraction of proteins with a Tris–HCl buffer containing sodium dodecyl sulphate (SDS) and dithiothreitol (DTT), precipitation of proteins with acetone, and solubilization in the Tris–HCl buffer. This methodology yielded the most successful isolation of pulp proteins and enabled the optimization of a UHPLC methodology for their separation. The method was applied to the profiling of olive pulp proteins from different olive cultivars observing in all cases a protein that had never been described before.     

Determination of Anionic Surfactant Turkey Red Oil by Capillary Electrophoresis with Direct UV Detection

A method using capillary electrophoresis with direct UV detection has been developed and validated for the determination of Turkey Red Oil (sulfonated castor oil). The highest performance with respect to separation efficiency and analysis time was achieved with 30 mM Tris (pH 8.0) buffer containing 7.5 mM HP-β-CD. The feasibility of the proposed CE method for the analysis of Turkey Red Oil surfactant in industrial water samples is demonstrated. Spiking of real samples gave recoveries between 90 and 106%. The CE results were compared with that obtained by GC-MS. It was concluded that CE can be a good alternative for fast determination of Turkey Red Oil component distribution in industrial process waters with no sample preparation other than dilution. However, the method sensitivity is not satisfactory for monitoring surfactant level in a waste effluent stream.     

Co-electroosmotic capillary electrophoresis determination of phenolic acids in commercial olive oil

Fourteen phenolic acids have been selectively determined in olive-oil samples using the co-electrosmotic capillary electrophoresis mode with UV detection after the LLE extraction system. A polycationic surfactant (hexadimetrine bromide, HDB), which dynamically coats the inner surface of the capillary and causes a fast anodic electroosmotic flow, was added to the electrolyte. The main factors affecting co-electroosmotic flow (EOF) such as type of modifier, concentration, and influence of organic solvents have been studied. Other parameters such as pH, type, and concentration of buffer, applied voltage, and injection time were also optimised using hydrodynamic injection for 8 s and UV detection at 210 nm. The composition optimum of the running buffer used was a 20% 2-propanol, 0.001% HDB, and 50 mM sodium borate at a pH value of 9.6. The method has been applied to determination and quantification of fourteen phenolic acids at ppb levels in olive oil samples after a liquid-liquid extraction.     

Extraction-liquid chromatography with electrochemical and spectrophotometric detection for the determination of copper and iron in biological and river water samples

Reversed-phase liquid chromatography using cupferron as a precolumn derivatizing agent was developed for the determination of Cu(II) and Fe(III) in biological materials and natural water samples. In the direct method, the metal cupferronates formed in acetonitrile-water (1 + 1) are injected onto an ODS column followed by separation with a mobile phase containing acetonitrile-acetate buffer (pH 3.5) (7 + 3) and other reagents. Amperometric detection with a glassy carbon electrode at ?0.40 V vs. Ag/AgCl can be used to determine both metals simultaneously. The electrochemical detection method has better sensitivity for the determination of Fe(III) than the usual spectrophotometric detection at 375 nm. If a large volume of aqueous sample is available, concentration of the two metal ions can be made by extraction with ethyl acetate prior to the chromatographic determination. In this case, liquid chromatographic separation and determination can be performed with the ODS column using a mobile phase consisting of acetonitrile-methanol-ethyl acetate-0.02 M acetate buffer (pH 3.5) (45 + 20 + 5 + 30).     

Analysis of the Amino Acids of Soy Globulins by AOT Reverse Micelles and Aqueous Buffer

The 7S and 11S globulins from soybean proteins using reverse micelle and aqueous buffer extraction methods were characterized by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscope (SEM), and their amino acid compositions were also evaluated. SDS-PAGE did not show electrophoretic differences between 7S and 11S globulin subunits with two extraction methods. SEM analysis showed that the AOT reverse micelle processing of 7S and 11S globulins induced a reduction of droplet size. Some individual amino acid contents of 7S and 11S globulins using two extraction methods were different, some were similar. In all the samples, the glutamic acid, aspartic acid, and leucine were the dominant amino acids while the cystine and methionine were the first-limiting amino acids. The proportion of essential amino acids to the total amino acids (E/T) of the 7S globulin from aqueous buffer and reverse micelles was similar. While significant differences were obtained in the proportion of E/T of the 11S globulin.