Rapid determination of aflatoxins in corn and peanuts

A rapid and simple method using ultra-high-pressure liquid chromatography with UV detection for the determination of aflatoxins B1, B2, G1 and G2 in corn and peanuts has been developed. In this method, aflatoxins were extracted with a mixture of acetonitrile and water (86:14) and then purified by solid-phase clean-up with a MycoSep#226 AflaZon(+) column. The toxins were determined by UPLC-UV without derivatizing aflatoxins in real samples, which has not been used in other studies. The mean recoveries of aflatoxins from non-infected peanut and corn samples spiked with aflatoxins B1, B2, G1 and G2 at concentrations from 0.22 to 5 microg/kg were between 83.4% and 94.7%. The detection limits (S/N=3) for B1, B2, G1 and G2 were 0.32, 0.19, 0.32 and 0.19 microg/kg, and the corresponding quantification limits (S/N=10) were 1.07, 0.63, 1.07 and 0.63 microg/kg, respectively. We also applied this method on real samples. Among 16 peanut samples, 2 (12.5% incidence) were contaminated with aflatoxin; among 18 corn samples, 4 (22% incidence) were contaminated. The proposed method is rapid, simple and accurate for monitoring aflatoxins in corn and peanuts.     

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.     

Cleanup procedure for determination of aflatoxins in major agricultural commodities by liquid chromatography

A simple, fast, reliable, and inexpensive chemical cleanup procedure was developed for quantitation of aflatoxins in major important agricultural commodities by liquid chromatography (LC). Aflatoxins were extracted from a ground sample with methanol-water (80 + 20, v/v), and after a single cleanup step on a minicolumn packed with basic aluminum oxide, they were quantitated by LC equipped with a C18 column, photochemical reactor, and fluorescence detector. Water-methanol-1-butanol (1,400 + 720 + 25, v/v/v) served as the mobile phase. Recoveries of aflatoxins B1, B2, G1, and G2 from peanuts spiked at 5.0, 2.5, 7.5, and 2.5 microg/kg were 87.2 +/- 2.3, 82.0 +/- 0.8, 80.0 +/- 1.8, and 80.4 +/- 2.8%, respectively. Similar recoveries, precision, and accuracy were achieved for corn, cottonseed, almonds, Brazil nuts, pistachios, and walnuts. The quantitation limit for aflatoxin B1 was 1 microg/kg. The minimal cost of the minicolumn allows for substantial savings compared with available commercial aflatoxin cleanup devices.     

Rapid immunoaffinity-based method for determination of zearalenone in corn by fluorometry and liquid chromatography

An immunoaffinity-based method was developed to determine zearalenone in corn. Corn samples were extracted in acetonitrile-water (90 + 10, v/v), applied to an immunoaffinity column, and eluted with methanol. The isolated toxin was quantitated either by reaction with aluminum chloride hexahydrate (AlCl3.6H2O) prior to measurement with a fluorometer or injection into a liquid chromatographic (LC) system with a fluorescence detector. Performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity, and comparison with AOAC Official Method 985.18. With the immunoaffinity column cleanup procedure, only zearalenone and its metabolites were recognized by the antibody (> or = 75% recovery). Limits of detection were 0.10 microgram/g for the fluorometer and 0.10 or 0.0025 microgram/g (sensitive method) for the LC method. Percentage recovery averaged 105% (fluorometer) and 93% (LC method), with average relative standard deviations (RSDs) of 15.7 and 9.3%. Naturally contaminated samples gave comparable RSDs of 8.3 and 9.9% for the fluorometer and LC methods, respectively. Column capacity was 4.0 micrograms with 89% recovery. Assay linearity was comparable for both methods (r2 = 0.998). Optimum assay ranges were 0.10-5.0 micrograms/g for the fluorometer and 0.10-50 or 0.0025-5.0 micrograms/g (sensitive method) for the LC method. Comparative analysis of 17 naturally contaminated corn samples using Zearala Test LC and the official AOAC LC method for detection of zearalenone showed that Zearala Test is statistically comparable to the AOAC Official Method 985.18 (r2 = 0.747).     

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.     

Determination of aflatoxins in peanuts by matrix solid-phase dispersion and liquid chromatography

A new method based on matrix solid-phase dispersion (MSPD) extraction was studied to determine aflatoxin B1, B2, G1 and G2 from peanuts. Optimization of different parameters, such as type of solid supports for matrix dispersion and elution solvents were carried out. The method used 2 g of peanut sample, 2 g of C18 bonded silica as MSPD sorbent and acetonitrile as eluting solvent. Recoveries of each aflatoxin spiked to peanut samples at 2.5 ng/g (5 ng/g for aflatoxin G2) level were between 78 and 86% with relative standard deviations ranging from 4 to 7%. The limits of quantification ranged from 0.125 to 2.5 ng/g for the four studied aflatoxins using liquid chromatography (LC) with fluorescence detection. In addition, LC coupled to mass spectrometry with an electrospray interface was used for confirmation of aflatoxins present in real samples. Eleven peanut samples from different countries were analyzed by the proposed method and by using an enzyme-linked immunosorbent assay (ELISA). ELISA test is a good screening method for investigation of these mycotoxins in peanut samples.     

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.     

Predicting aflatoxin and fumonisin in shelled corn lots using poor-quality grade components

A study was conducted to determine if aflatoxin and fumonisin are concentrated in the poor-quality grade components of shelled corn. Four 1.0 kg test samples were each taken from 23 lots of shelled corn marketed in North Carolina. Inspectors from the Federal Grain Inspection Service divided each test sample into 3 grade components: (1) damaged kernels (DM), (2) broken corn and foreign material (BCFM), and )3) whole kernels (WH). The aflatoxin and fumonisin concentration was measured in each component and a mass balance equation was used to calculate the total concentration of each mycotoxin in each test sample. Averaged across all test samples, the aflatoxin concentrations in the DM, BCFM, and WH components were 1300.3, 455.2, and 37.3 ppb, respectively. Averaged across all test samples, the fumonisin concentrations in the DM, BCFM, and WH components were 148.3, 51.3, and 1.8 ppm, respectively. The DM and BCFM components combined accounted for only 5.0% of the test sample mass, but accounted for 59.8 and 77.5% of the total aflatoxin and fumonisin mass in the test sample, respectively. Both aflatoxin mass (ng) and aflatoxin concentration (ng/g) in the combined DM and BCFM components had high correlations with aflatoxin concentration in the lot. The highest correlation occurred when aflatoxin mass (ng) in the combined DM and BCFM components was related to aflatoxin concentration in the lot (0.964). Similar results were obtained for fumonisin. This study indicated that measuring either aflatoxin or fumonisin in the combined DM and BCFM grade components could be used as a screening method to predict either aflatoxin or fumonisin in a bulk lot of shelled corn.     

Determination of type B fumonisin mycotoxins in maize and maize-based products by liquid chromatography/tandem mass spectrometry using a QqQlinear ion trap mass spectrometer

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determining the type B fumonisin mycotoxins in corn-based foodstuffs is described. Fumonisins FB1 and FB2 were extracted from a 1 g sample by homogenization with acetonitrile/water (75:25, v/v, 50 mmol/L formic acid, 25 mL final volume) and the extract was defatted on C18 phase. Volumes of 5 mL of crude extracts were cleaned up on Carbograph-4 cartridges. The final solution was analyzed by HPLC with electrospray ionization mass spectrometry in positive ion mode using multiple reaction monitoring with a QqQ linear ion trap mass spectrometer. Recoveries for spiked corn-based foodstuffs ranged from 91-105% (RSD% < or =8%), and method detection limits were < or =2 ng/g for FB1 and < or =1 ng/g for FB2. Two different spiking levels were tested (5000 and 100 ng/g for FB1, 1000 and 20 ng/g for FB2). Quantitation was achieved by an external calibration procedure using matrix-matched standards, with diclofenac added post-cleanup as internal standard for the LC/MS/MS analyses. Calibration curves showed linearity in the concentration range 0.005-5 ng/microL of final extract (0.992 < or = R2< or =0.995). Two other fumonisins, FB3 and FB4, were identified in naturally contaminated samples of corn meal using an information-dependent acquisition protocol that looped three experiments, including neutral loss scan, enhanced resolution scan, and enhanced product ion scan. FB3 and FB4 quantitation was estimated as peak area ratios relative to the FB2 response in view of the lack of both standards. This work also includes an application of the present LC/MS/MS method to some maize and maize-based product samples (corn meal, cornflakes and popcorn) collected from Italian stores. FB1 and FB2 contamination levels exceeding the European Union recommendation were found in 8 out of 15 corn meal samples.     

Aflatoxin plate kit. Performance Tested Method 081003

The level of total aflatoxin contamination was analyzed in naturally contaminated and spiked samples of corn and peanut using the Aflatoxin Plate Kit. This kit is an enzyme-linked immunosorbent assay (ELISA) suitable for rapid testing of grains and peanuts. The assay was evaluated for ruggedness and linearity of the standard curve. The test kit results were then statistically evaluated for accuracy, precision, and correlation to a validated HPLC method (AOAC 994.08). The results were verified by an independent laboratory.     

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin B1 in corn samples: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 levels in corn samples, enforced by European Union legislation. A test portion was extracted with methanol-water (80 + 20); the extract was filtered, diluted with phosphate-buffered saline solution, filtered on a microfiber glass filter, and applied to an immunoaffinity column. The column was washed with deionized water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase LC with fluorescence detection after either pre- or postcolumn derivatization. Precolumn derivatization was achieved by generating the trifluoroacetic acid derivative, used by 8 laboratories. The postcolumn derivatization was achieved either with pyridinium hydrobromide perbromide, used by 16 laboratories, or with an electrochemical cell by the addition of bromide to the mobile phase, used by 5 laboratories. The derivatization techniques used were not significantly different when compared by the Student's t-test; the method was statistically evaluated for all the laboratories. Five corn sample materials, both spiked and naturally contaminated, were sent to 29 laboratories (22 Italian and 7 European). Test portions were spiked with aflatoxin B1 at levels of 2.00 and 5.00 ng/g. The mean values for recovery were 82% for the low level and 84% for the high contamination level. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the values for relative standard deviation for repeatability (RSDr) ranged from 9.9 to 28.7%. The values for relative standard deviation for reproducibility (RSDR) ranged from 18.6 to 36.8%. The method demonstrated acceptable within- and between-laboratory precision for this matrix, as evidenced by the HorRat values.     

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.     

在线热辅助甲基化-气相色谱法分析棉籽仁中的脂肪酸组成

建立了分析棉籽仁中脂肪酸组成的在线热辅助甲基化-气相色谱法。将0.3 mg棉籽仁样品与2 μ L三甲基氢氧化硫（0.2 mol/L）加入裂解器,在350℃下进行甲基化反应,通过气相色谱仪进行分离分析,共检测到8种脂肪酸甲酯成分,分别为亚油酸（C18：2）、油酸（C18：1）、棕榈酸（C16：0）、硬脂酸（C18：0）、肉豆蔻酸（C14：0）、棕榈油酸（C16：1）、花生酸（C20：0）和二十二酸（C22：0）,不饱和脂肪酸的相对含量为66.30%~72.54%,其中亚油酸的相对含量为43.20%~53.61%,相对峰面积的相对标准偏差（RSD）小于10%（n=5）。通过分析5组棉籽仁样品与3种食用油中的脂肪酸组成,结果表明不同产地的棉籽仁中的脂肪酸组成差异不明显,且棉籽仁中的脂肪酸组成与玉米油最为接近,相似度为0.960~0.992。该方法简单、快速、准确,适合分析棉籽仁中的脂肪酸组成。     

高效液相色谱-串联质谱法检测花生中的黄曲霉毒素B_1

应用高效液相色谱-电喷雾串联四极杆质谱联用系统(HPLC-MS/MS),在多反应离子检测方式(MRM)下,对花生中的黄曲霉毒素B1进行检测.对花生中黄曲霉毒素B1的提取、净化、液相分离及串联质谱等相关检测参数进行了优化研究.用V(甲醇):V(水)=6:4提取,OASIS HLB SPE小柱净化,定容过滤.采用V(甲醇):V(水)(含体积分数0.1%甲酸)=7:3为流动相,前级离子313.0,二级离子241.1、269.1,ESI正离子方式检测,在3.3 min出峰.结果表明,在ESI正离子模式下,黄曲霉毒素B1在其线性定量范围0.1～50μg/kg内,相关系数达到0.9999,检出限为0.03μg/kg,最低定量限为0.1μg/kg.低、中、高浓度添加回收率范围为93%～105%.     

Excitation-emission matrix fluorescence spectroscopy in conjunction with multiway analysis for PAH detection in complex matrices

A field portable, single exposure excitation-emission matrix (EEM) fluorometer has been constructed and used in conjunction with parallel factor analysis (PARAFAC) to determine the sub part per billion (ppb) concentrations of several aqueous polycyclic aromatic hydrocarbons (PAHs), such as benzo(k)fluoranthene and benzo(a)pyrene, in various matrices including aqueous motor oil extract and asphalt leachate. Multiway methods like PARAFAC are essential to resolve the analyte signature from the ubiquitous background in environmental samples. With multiway data and PARAFAC analysis it is shown that reliable concentration determinations can be achieved with minimal standards in spite of the large convoluting fluorescence background signal. Thus, rapid fieldable EEM analyses may prove to be a good screening method for tracking pollutants and prioritizing sampling and analysis by more complete but time consuming and labor intensive EPA methods.     

Testing shelled corn for aflatoxin, Part II: modeling the observed distribution of aflatoxin test results

The suitability of several theoretical distributions to predict the observed distribution of aflatoxin test results in shelled corn was investigated. Fifteen positively skewed theoretical distributions were each fitted to 18 empirical distributions of aflatoxin test results for shelled corn. The compound gamma distribution was selected to model aflatoxin test results for shelled corn. The method of moments technique was chosen to estimate the parameters of the compound gamma distribution. Mathematical expressions were developed to calculate the parameters of the compound gamma distribution for any lot aflatoxin concentration and test procedure. Observed acceptance probabilities were compared to operating characteristic curves predicted from the compound gamma distribution, and all 18 observed acceptance probabilities were found to lie within a 95% confidence band. The parameters of compound gamma were used to calculate the fraction of aflatoxin-contaminated kernels in contaminated lots. At 20 ppb, it was estimated that about 6 in 10,000 kernels are contaminated.     

Simultaneous quantitation of Aspergillus flavus/A. parasiticus and aflatoxins in peanuts

A method was developed for simultaneous quantitation of Aspergillus flavus/A. parasiticus and aflatoxins in peanuts. Peanut samples were ground with an equal weight of water in a vertical cutter mixer to produce a slurry. Separate subsamples were taken for dilution-plating to determine total colony forming units (CFU)/g of A. flavus/A. parasiticus and for liquid chromatographic analysis to determine aflatoxin concentrations. Dry-grinding peanuts for homogenization of aflatoxins produced high temperatures that killed most of the A. flavus/A. parasiticus propagules. Addition of water to produce a slurry kept the temperature from rising above levels that killed the fungi. A 7 min grind time provided optimal homogenization for both the fungi and aflatoxins, so long as the temperature of the slurry did not exceed 45 degrees C. In the analysis of 60 shelled peanut samples, total aflatoxin concentrations ranged from 0 to 10,000 ng/g and total A. flavus/A. parasiticus ranged from 1.4 x 10(3) to 3.2 x 10(6) CFU/g. Regression analysis showed a significant positive correlation (p < 0.0001) between the quantities of A. flavus/A. parasiticus and aflatoxin (R2 = 0.82).     

Testing shelled corn for aflatoxin, Part I: estimation of variance components

The variability associated with testing lots of shelled corn for aflatoxin was investigated. Eighteen lots of shelled corn were tested for aflatoxin contamination. The total variance associated with testing shelled corn was estimated and partitioned into sampling, sample preparation, and analytical variances. All variances increased as aflatoxin concentration increased. With the use of regression analysis, mathematical expressions were developed to model the relationship between aflatoxin concentration and the total, sampling, sample preparation, and analytical variances. The expressions for these relationships were used to estimate the variance for any sample size, subsample size, and number of analyses for a specific aflatoxin concentration. Test results on a lot with 20 parts per billion aflatoxin using a 1.13 kg sample, a Romer mill, 50 g subsamples, and liquid chromatographic analysis showed that the total, sampling, sample preparation, and analytical variances were 274.9 (CV = 82.9%), 214.0 (CV = 73.1 %), 56.3 (CV = 37.5%), and 4.6 (CV = 10.7%), respectively. The percentage of the total variance for sampling, sample preparation, and analytical was 77.8, 20.5, and 1.7, respectively.     

A sensitive confirmatory method for aflatoxins in maize based on liquid chromatography/electrospray ionization tandem mass spectrometry

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for measurement of aflatoxins B1, B2, G1, and G2 in maize is described. Aflatoxins (AFs) were extracted from 1 g samples by using tri-portions of acetonitrile/water (80:20, v/v) (10 + 7 + 7 mL), and 2/5 of the extract diluted to 500 mL by water was cleaned up with a 100 mg Carbograph-4 cartridge. After the addition of the internal standard AFM1, the final extract was analyzed by LC/ESI-MS/MS in positive ion mode using multiple reaction monitoring with a triple-quadrupole instrument. A C(18) column thermostatted at 45 degrees C with a mobile phase gradient of acetonitrile/water with 2 mmol/L ammonium formate was used. Although the matrix suppression effect was negligible, quantitation was achieved by an external calibration procedure using matrix-matched standard solutions to improve accuracy. Sample recoveries at four spiking levels ranged from 81 to 101% (relative standard deviation (RSD) 相似文献