Simultaneous determination of aflatoxins B2 and G2 in peanuts using spectrofluorescence coupled with parallel factor analysis

In the present study a method for the simultaneous determination of aflatoxins B₂ and G₂ in peanuts has been developed. The method uses second order standard addition method and excitation–emission fluorescence data together with parallel factor analysis (PARAFAC). The aflatoxin analysis was based on extraction with methanol–water and carried out using immunoaffinity clean-up. The results of PARAFAC on a set of spiked and naturally contaminated peanuts indicated that the two aflatoxins could be successfully determined. The method was validated and analytical figures of merit were obtained for both analytes. The limits of detection (LOD) were 0.05 and 0.04 μg kg⁻¹ for aflatoxins B₂ and G₂, respectively. The limits of quantification (LOQ) were 0.16 and 0.12 μg kg⁻¹ for aflatoxins B₂ and G₂, respectively. Coupling of spectrofluorimetry with PARAFAC can be considered as an alternative method for quantification of aflatoxins in the presence of unknown interferences obtained through analysis of highly complex matrix of peanuts samples at a reduced cost per analysis.     

Continuous production of butanol from starch-based packing peanuts

Acetone, butanol, ethanol (ABE, or solvents) were produced from starch-based packing peanuts in batch and continuous reactors. In a batch reactor, 18.9 g/L of total ABE was produced from 80 g/L packing peanuts in 110 h of fermentation. The initial and final starch concentrations were 69.6 and 11.1 g/L, respectively. In this fermentation, ABE yield and productivity of 0.32 and 0.17 g/(L·h) were obtained, respectively. Compared to the batch fermentation, continuous fermentation of 40 g/L of starch-based packing peanuts in P2 medium resulted in a maximum solvent production of 8.4 g/L at a dilution rate of 0.033 h⁻¹. This resulted in a productivity of 0.27 g/(L·h). However, the reactor was not stable and fermentation deteriorated with time. Continuous fermentation of 35 g/L of starch solution resulted in a similar performance. These studies were performed in a vertical column reactor using Clostridium beijerinckii BA101 and P2 medium. It is anticipated that prolonged exposure of culture to acrylamide, which is formed during boiling/autoclaving of starch, affects the fermentation negatively.     

Simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products by ultra-high-performance liquid chromatography-tandem mass spectrometry

A reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products was developed. The sample was extracted by 84% of acetonitrile aqueous solution and the extract was purified by a reliable solid phase extraction-based clean-up method. Then, the analytes were separated on Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm particle size), and eluted with a mobile phase consisting of (A) water containing 0.1% formic acid and (B) acetonitrile/methanol (50/50, v/v). The separated compounds were detected with a Waters Micromass Quattro Ultima Pt tandem quadrupole mass spectrometer operating in positive electro-spray ionization using multiple reaction monitoring mode. The established method was extensively validated by determining the linearity (R² ≥ 0.9990), average recovery (74.7-86.8%) and precision (relative standard deviation ≤ 10.9%). It was shown to be a suitable method for simultaneous determination of the six aflatoxins in peanuts and their derivative products. Finally, a total of 73 samples randomly collected from different areas in Zhejiang province were screened for aflatoxins with the proposed method. The results showed that 31 samples of peanut butter, 14 samples of fresh peanut and 5 samples of musty peanut were contaminated with aflatoxins. Meanwhile, this was the first report on aflatoxins M1 and M2, which were found in unprocessed peanuts and their derivative products.     

Development and validation of a (semi-)quantitative UHPLC-MS/MS method for the determination of 191 mycotoxins and other fungal metabolites in almonds, hazelnuts, peanuts and pistachios

A multi-target method for the determination of 191 fungal metabolites in almonds, hazelnuts, peanuts and pistachios was developed. The method includes all mycotoxins regulated in the European Union and mycotoxins regularly found in food. After extraction with an acidified acetonitrile water mixture, the raw extract was diluted and injected directly into the UHPLC-MS/MS system. In two chromatographic runs, analysis was performed in positive and in negative ionisation mode. The method was in-house validated for the most important 65 analytes in these four commodities. Apparent recoveries between 80 and 120 % were obtained for about half of the analyte–matrix combinations. Good repeatabilities (standard deviations?<?10 %) were achieved for the vast majority (83 %) of all cases. Only in 6 % of all combinations did the standard deviations exceed 15 %. Matrix effects, arising during electrospray ionisation, significantly influenced the determination. For instance, signal suppression was observed for several early-eluting analytes and also signal enhancement up to 295 % for physcion in peanuts was determined. Concerning extraction recovery, 94 % of the analyte–matrix combinations showed values higher than 50 %. Lower limits of quantification ranged between 0.04 μg?kg^?1 for enniatin B3 in peanuts and 500 μg?kg^?1 for HC toxin in hazelnuts. Additionally, the applicability of the developed method was demonstrated through the analysis of 53 naturally contaminated nut samples from Austria and Turkey. Overall, 40 toxins were quantified; the most frequently found mycotoxins were beauvericin (79 %), enniatin B (62 %) and macrosporin (57 %). In the most contaminated hazelnut sample, 26 different fungal metabolites were detected.     

Laboratory proficiency testing of aflatoxins in corn and peanuts--a cooperative project between Thailand and the United States

The objective of this project was to conduct an aflatoxin proficiency test program in government, academia, and industry laboratories in Thailand. Aflatoxin-free corn and peanuts and corn and peanuts naturally contaminated with aflatoxins diluted to approximately 25 micrograms/kg were analyzed. Homogeneity of prepared, naturally contaminated test samples was checked on multiple replicates. The test was conducted according to the ISO/IUPAC/AOAC INTERNATIONAL Harmonized Protocol with z scores indicating laboratory performance. The participants used 3 methods: enzyme-linked immunosorbent assay, thin-layer chromatography, and the minicolumn. Of 19 laboratories that reported results for aflatoxins in naturally contaminated corn, 13 (68%) performed satisfactorily, on the basis of the mean obtained by an expert laboratory, a calculated target value for standard deviation, and the z score. Of 21 laboratories that reported results for aflatoxins in naturally contaminated peanuts, 10 (48%) performed satisfactorily. For aflatoxin-free corn, 6 laboratories reported finding aflatoxins at > or = 10 ng/g, chiefly by the minicolumn method; for aflatoxin-free peanuts, 1 laboratory reported finding aflatoxins at > 10 ng/g. Subsequently, a workshop of lectures and laboratory sessions was conducted to improve performance. A new and simple successive outlier removal procedure applied to the same data removed the same laboratories as did the use of z scores.     

Optimized Ultrasonic-Assisted Extraction of Aflatoxin B1 in Peanuts with Response Surface Methodology

Response surface methodology was used to optimize the ultrasonic-assisted extraction conditions for aflatoxin B1 in peanuts. Box–Behnken center composite optimization was performed with factors including the ratio of sample to extractant, sonication time, and sonication temperature. Response surface methodology was used with three factors and three levels to determine the prime factors on the extraction of aflatoxin B1. High-performance liquid chromatography with fluorescence detector was used for the determination of aflatoxin B1. The optimum conditions were 1?g peanuts to 86?mL extractant, a sonication time of 7?min, and a temperature of 73°C. The mean recoveries in fortified peanuts were between 87.6 and 93.5% with an interday and intraday relative standard deviations below 10.6 and 9.8%, respectively. The developed method was accurate and precise for the determination of aflatoxin B1.     

Molecular Modeling of Metabolism for Allergen-Free Low Linoleic Acid Peanuts

It is necessary to eliminate linoleic acid and allergenic arachins from peanuts for good health reasons. Virginia-type peanuts, harvested from plots treated with mineral salts combinations that mimic the subunit compositions of glutamate dehydrogenase (GDH) were analyzed for fatty acid and arachin compositions by HPLC and polyacrylamide gel electrophoresis, respectively. Fatty acid desaturase and arachin encoding mRNAs were analyzed by Northern hybridization using the homologous RNAs synthesized by peanut GDH as probes. There were 70?C80?% sequence similarities between the GDH-synthesized RNAs and the mRNAs encoding arachins, fatty acid desaturases, glutamate synthase, and nitrate reductase, which similarities induced permutation of the metabolic pathways at the mRNA level. Modeling of mRNAs showed there were 210, 3,150, 1,260, 2,520, and 4,200 metabolic permutations in the control, NPKS-, NS-, Pi-, NH₄Cl-, and PK-treated peanuts, respectively. The mRNA cross-talks decreased the arachin to almost zero percent in the NPKS- and PK-treated peanuts, and linoleate to ??18?% in the PK-treated peanut. The mRNA cross-talks may account for the vastly reported environmentally induced variability in the linoleate contents of peanut genotypes. These results have quantitatively unified molecular biology and metabolic pathways into one simple biotechnology for optimizing peanut quality and may encourage small-scale industry to produce arachin-free low linoleate peanuts.     

Validation of two commercial lateral flow devices for the detection of peanut proteins in cookies: interlaboratory study

Results are reported for an interlaboratory validation study of 2 commercially available Iateral flow devices (dipstick tests) designed to detect peanut residues in food matrixes. The test samples used in this study were cookies containing peanuts at 7 different concentrations in the range of 0-30 mg peanuts/kg food matrix. The test samples with sufficient and proven homogeneity were prepared in our laboratory. The analyses of the samples (5 times per level by each laboratory) were performed by 18 laboratories worldwide which submitted a total of 1260 analytical results. One laboratory was found to be an outlier for one of the test kits. In general, both test kits performed well. However, some false-negative results were reported for all matrixes containing < 21 mg peanuts/kg cookie. It must be stressed that the test kits were challenged beyond their cut-off limits (> or = 5 mg/kg, depending on the food matrix). One test kit showed fewer false-negative results, but it led to some false-positive results for the blank materials. The sensitivity of the dipstick tests approaches that achieved with enzyme-linked immunosorbent assays.     

Validation study of immunoaffinity column chromatography coupled with solution fluorometry or HPLC for the detection of aflatoxin in peanuts and corn

Neogen Corp. has developed the Neocolumn for Aflatoxin DR for the detection of total aflatoxin by HPLC or solution fluorometry. The purpose of this study was to validate the method under the requirements of the AOAC Research Institute Performance Tested Methods (PTM) program. There are several AOAC Official Methods for detection of total aflatoxin in corn; they consist of rapid and analytical-based methods and two rapid methods (PTMs 030701 and 050901) that have been performance tested by the AOAC Research Institute. A widely used reference method, however, is AOAC Official Method 991.31, which uses immumoaffinity cleanup followed by HPLC or solution fluorometry and is referred to as the reference method in this document. In internal studies, the Neocolumn method coupled with solution fluorometry demonstrated a relative recovery from peanuts of 101.6% of the reference value, with a CV of 3.9% across all levels analyzed; when coupled with HPLC, the Neocolumn method demonstrated a relative recovery from peanuts of 103.0% of the reference value with a CV of 3.5% across all levels analyzed. The Neocolumn method coupled with solution fluorometry demonstrated a relative recovery from corn of 116.9% of the reference value with a CV of 6.1% across all levels analyzed; when coupled with HPLC, the Neocolumn method demonstrated a relative recovery from corn of 91.2% of the reference value, with a CV of 5.4% across all levels analyzed. Calculations were made by comparison with the mean result obtained by the HPLC reference method, which showed respective CV values of 3.9 and 2.0% for recoveries from peanuts and corn, respectively.     

石墨炉原子吸收法测定黑花生中的硒

该文以转基因食品黑花生为研究对象,针对硒易挥发的特点,建立了高压密闭消解-石墨炉原子吸收光谱法测定黑花生中总硒含量的测定方法.研究表明,HNO3-H2O2可使样品达到最佳消解,以Pd(NO3)2+Mg(NO3)2为基体改进剂,最佳灰化温度和原子化温度分别为500 ℃和2 000 ℃.在优化实验条件下,该方法测定硒的线性...     

Determination of cyclopiazonic acid in food and feeds by liquid chromatography–tandem mass spectrometry

A new, fast and efficient multiple reaction monitoring (MRM) high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the determination of cyclopiazonic acid (CPA) in mixed feed, wheat, peanuts and rice is presented. The analytical methodology involves sample extraction with an alkaline methanol–water mixture, defatting with hexane and quantification using HPLC–MS/MS without further treatment of sample extracts. Reversed-phase liquid chromatography using a C18 stationary phase coupled to negative mode electrospray triple quadrupole tandem mass spectrometry was applied. The limit of detection was 5 μg/kg while the limit of quantification was 20 μg/kg in the matrices investigated. The detector response was found to be linear over the range 25–250 μg/kg in feed and 25–500 μg/kg in wheat, peanuts and rice. The mean overall recoveries (n = 18) of CPA varied from 79% to 114% in the range of concentrations studied over a period of 4 months. Mean recoveries (n = 3 or 6) of CPA in wheat, peanuts and rice varied from 70% to 111%, 77% to 116% and 69% to 92%, respectively. The method was successfully applied to the analysis of feed and rice samples artificially infected with the fungal strain Penicillium commune, where the toxin was found at different levels.     

Optimization of roasting process and product quality of peanuts

Differential scanning calorimetry (DSC) was used to establish criteria for optimization of raw material selection, roasting process, eating quality, visual appearance, and shelf-life extension of peanuts [1-4]. DSC methods were developed as both predictive and analytical tools to define process operating guidelines and to correlate with traditional quality attributes of roasted peanuts [1-4].     

Graft copolymerization of epichlorohydrin and ethylenediamine onto cellulose derived from agricultural by-products for adsorption of Pb(II) in aqueous solution

In order to develop a low-cost and high efficient absorbent, cellulose was extracted from peanut hulls, soybean shells and grapefruit peels using 17.5 % NaOH and then copolymerized with epichlorohydrin and ethylenediamine. Infrared spectra and N contents show that the cellulose was copolymerized successfully with the ethylenediamine. Factors affecting the adsorption behavior of Pb(II), such as pH, temperature, ratio of solid to liquid, competitive sorption of various metal ions, initial metal concentration and adsorption time, were then investigated. The adsorption equilibrium could be obtained within 120 min and the kinetic adsorption processes fitted well with the pseudo-second order kinetic model. The isotherm adsorption data fitted well with Langmuir adsorption model and the maximum absorption capacities of the modified peanut hulls, soybean shells and grapefruit peels were 47.8, 101 and 232 mg g^?1, respectively. The competitive adsorption of mixed metal ions demonstrated that Pb(II) was preferentially removed from solution by the modified peanuts shells, soybean shells and grapefruit peels, then Cu(II) and Cr(III). Desorption of Pb(II) from modified peanut hulls, soybean shells and grapefruit peels was effectively achieved in a 1 mol L^?1 HCl solution. Ethylenediamine-modified grapefruit peels exhibited higher absorption performance than the ethylenediamine-modified soybean shells and peanut hulls and can be used as potential low-cost and high efficient absorbents for the removal of lead ions from wastewater.     

Preparation of peanut butter suspension for determination of peanuts using enzyme-linked immunoassay kits

Peanuts are one of the 8 most common allergenic foods and a large proportion of peanut-allergic individuals have severe reactions, some to minimal exposure. Specific protein constituents in the peanuts are the cause of the allergic reactions in sensitized individuals who ingest the peanuts. To avoid accidental ingestion of peanut-contaminated food, methods of analysis for the determination of the allergenic proteins in foods are important tools. Such methods could help identify foods inadvertently contaminated with peanuts, thereby reducing the incidence of allergic reactions to peanuts. Commercial immunoassay kits are available but need study for method performance, which requires reference materials for within- and between-laboratory validations. In this study, National Institute of Standards and Technology Standard Reference Material 2387 peanut butter was used. A polytron homogenizer was used to prepare a homogenous aqueous Peanut Butter suspension for the evaluation of method performance of some commercially available immunoassay kits such as Veratox for Peanut Allergen Test (Neogen Corp.), Ridascreen Peanut (R-Biopharm GmbH), and Bio-Kit Peanut Protein Assay Kit (Tepnel). Each gram of the aqueous peanut butter suspension contained 20 mg carboxymethylcellulose sodium salt, 643 microg peanut, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. The suspension was homogenous, stable, reproducible, and applicable for adding to ice cream, cookies, breakfast cereals, and chocolate for recovery studies at spike levels ranging from 12 to 90 microg/g.     

Liquid chromatographic analysis of aflatoxin using post-column photochemical derivatization: collaborative study

Aflatoxin analysis, with post-column derivatization using a photochemical reactor for enhanced detection (PHRED) system for derivatization, has been compared to the officially recognized iodine and Kobra cell derivatization systems. This photochemical system has been extensively used for screening peanuts by some U.S. Department of Agriculture laboratories for many years. From their periodic method checks, using standard spiked samples, an 80 sample series with each of the 3 derivatization methods was statistically analyzed. Paired comparisons, using the same sample extract, were also made between the PHRED and one of the other 2 methods, among laboratories in 4 different countries, on a variety of naturally contaminated commodity products. The differences between the techniques were not significant for peanuts, but for corn the photochemical system consistently gave slightly higher values for aflatoxins B1 and B2 than the Kobra cell method. However, a comparison of all sample results showed no significant differences between methods. The Pearson correlation coefficients for aflatoxin B1 in 102 test samples and aflatoxin B2 in 94 test samples were 0.9994 and 0.9874, respectively. The probability factor was P < 0.0001, and the t-tests were not significantly different except for the corn. These indicated that the PHRED system is equivalent to the iodine and Kobra cell methods for peanuts relative to the current official procedures, but the PHRED system has a slightly high bias for corn compared to the iodine and Kobra cell systems.     

Properties of Ozone-Oxidized Tapioca Starch and Its Use in Coating of Fried Peanuts

Oxidation of tapioca via ozone oxidation was carried out under different conditions in comparison with H₂O₂. The impact of ozonation on physicochemical properties of tapioca was studied and fried peanuts coated with different tapioca were characterized. Different ozone oxidation times (10, 20, and 30 min) and various pH values (5, 7, and 9) were used for tapioca modification. Tapioca oxidized by ozone for 20 min at pH 7 had higher swelling power (SP), water holding capacity (WHC), oil holding capacity (OHC), and viscosity than the native counterpart (P < 0.05). This coincided with the higher carbonyl and carboxyl contents (P < 0.05). The highest frying expansion (FE) with the lowest hardness was attained for fried peanut coated with tapioca oxidized under the aforementioned condition. Therefore, oxidation of tapioca using ozone under optimal conditions could be a potential means to improve frying expansion as well as the crispiness of the fried coated peanuts.     

Purification of phospholipase D by two-phase affinity extraction

An aqueous two-phase system of polyethylene glycol (PEG)-salt was used for purification of phospholipase D (PLD) from peanuts and carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using 0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.     

Competitive inhibition ELISA for quantification of Ara h 1 and Ara h 2, the major allergens of peanuts

Allergies to peanuts are becoming an increasingly important health problem as a result of the persistence and severity of the reaction in allergic individuals. Because no treatment currently is available, avoidance is the only option for peanut-allergic individuals. Avoidance of an abundant and often disguised food such as peanuts, however, is very difficult; therefore, competitive inhibition ELISAs were developed to detect and quantitate each of the major peanut allergens, Ara h 1 and Ara h 2. Under optimal conditions for each assay, the sensitivity of the Ara h 1 and Ara h 2 detection assays were 12 and 0.5 ng/mL, respectively. These assays were primarily devised to effectively compare the levels of Ara h 1 and Ara h 2 in a wide variety of peanuts or peanut products but can also be used to identify cross-reactive antigens. The method is simple and rapid, requiring only one allergen-specific antibody and, therefore, could be adapted specifically to detect the presence of these individual allergens in different foods.     

Variation of analytical results for peanuts in energy bars and milk chocolate

Peanuts contain proteins that can cause severe allergic reactions in some sensitized individuals. Studies were conducted to determine the percentage of recovery by an enzyme-linked immunosorbent assay (ELISA) method in the analysis for peanuts in energy bars and milk chocolate and to determine the sampling, subsampling, and analytical variances associated with testing energy bars and milk chocolate for peanuts. Food products containing chocolate were selected because their composition makes sample preparation for subsampling difficult. Peanut-contaminated energy bars, noncontaminated energy bars, incurred milk chocolate containing known levels of peanuts, and peanut-free milk chocolate were used. A commercially available ELISA kit was used for analysis. The sampling, sample preparation, and analytical variances associated with each step of the test procedure to measure peanut protein were determined for energy bars. The sample preparation and analytical variances were determined for milk chocolate. Variances were found to be functions of peanut concentration. Sampling and subsampling variability associated with energy bars accounted for 96.6% of the total testing variability. Subsampling variability associated with powdered milk chocolate accounted for >60% of the total testing variability. The variability among peanut test results can be reduced by increasing sample size, subsample size, and number of analyses. For energy bars the effect of increasing sample size from 1 to 4 bars, subsample size from 5 to 20 g, and number of aliquots quantified from 1 to 2 on reducing the sampling, sample preparation, and analytical variance was demonstrated. For powdered milk chocolate, the effects of increasing subsample size from 5 to 20 g and number of aliquots quantified from 1 to 2 on reducing sample preparation and analytical variances were demonstrated. This study serves as a template for application to other foods, and for extrapolation to different sizes of samples and subsamples as well as numbers of analyses.     

气相色谱法测定花生中六六六、滴滴涕、乙草胺和毒死蜱残留

建立了毛细管柱气相色谱法同时测定花生中六六六、滴滴涕、乙草胺和毒死蜱的方法。样品用乙腈提取,固相萃取柱净化,气相色谱仪电子捕获检测器(ECD)进行测定。结果表明,10种农药含量在0.005~1.0 mg/L之间线性关系良好,相关系数均在0.995以上,加标回收率在80.5%~116.9%之间,相对标准偏差在1.2%~5.0%之间。方法适用于花生中六六六、滴滴涕、乙草胺和毒死蜱的同时测定。