Determination of aflatoxin concentrations in peanut butter by enzyme-linked immunosorbent assay (ELISA): study of three commercial ELISA kits

Sixteen United Kingdom analytical laboratories participated in an evaluation of 3 commercially available enzyme-linked immunosorbent assay (ELISA) kits for analysis of aflatoxin in peanut butter. Each laboratory was sent 3 sets of 10 randomly numbered samples of peanut butter. Each set consisted of 5 pairs of undisclosed duplicates. Four of the sets of duplicates were naturally contaminated butters with "target" aflatoxin values (estimated by liquid chromatography) between 8 and 81 micrograms/kg. The fifth pair was a blank peanut butter containing approximately 3 micrograms/kg of total aflatoxins. A statistical treatment of the results of the study is presented, together with discussion of the relative merits of the different kits.     

High pressure liquid chromatographic determination of aflatoxins in peanut butter using a silica gel-packed flowcell for fluorescence detection

A high pressure liquid chromatographic method has been developed for determining aflatoxins B1, B2, G1, and G2 in peanut butter. The method is based on extraction with acidified aqueous methanol, partition of the aflatoxin into methylene chloride, and purification of the extract on a 2 g silica gel column. The extracted aflatoxins are resolved on a microparticulate (10 micrometer) porous silica gel column in ca 10 min with a water-washed chloroform-cyclohexane-acetonitrile solvent that contains 2% isopropanol. The fluorescence detection system determines aflatoxins B1, B2, G1, and G2 at low levels, i.e., 0.25 ppb B1, 0.5 ppb G1, and 0.2 ppb B2 and G2. Multiple assays of 5 samples of naturally contaminated peanut butters containing total aflatoxins (B1 + B2 + G1 + G2) at levels of 1, 2, 3, 9, and 17 ppb gave intralaboratory coefficients of variation of 7, 4, 4, 11, and 3%, respectively. Samples spiked at levels of 5, 9, and 17 ppb total aflatoxins showed recoveries of 79, 81, and 81%, respectively.     

Development of a fluorescence polarization assay for the determination of aflatoxins in grains

Aflatoxins produced by Aspergillus flavus are commonly found in human and animal foods including grains, cereals, peanut products, sorghum, and soy seeds. Exposure to aflatoxins has been associated with carcinogenicity. This paper reports a simple, portable, and rapid fluorescence polarization (FP) assay for aflatoxin determination in grains. This immunoassay is field portable, homogeneous, and without any washing and cleaning steps. The assay is based upon the competition between free aflatoxin and an aflatoxin-fluorescein tracer for an aflatoxin-specific monoclonal antibody in solution. A series of naturally contaminated corn, sorghum, peanut butter, and peanut paste samples were analyzed by FP and compared with HPLC results. Similarly, spiked popcorn samples were analyzed by FP. FP results of naturally contaminated samples correlated well with HPLC (r (2) = 0.97). FP analysis of spiked popcorn samples (with a mixture of B(1)/B(2)/G(1)/G(2), 7/1/3/1, w/w) gave a good correlation with spiked values (r (2) = 0.99). However, FP consistently underestimated the aflatoxin contents. This was perhaps due to low cross-reactivity of the antibody used toward B(2), G(1), and G(2) aflatoxins. These results combined with the portability and simplicity of the assay suggest that the assay can be used for screening total aflatoxin in grains.     

Non-detectable levels of trans-fatty acids in peanut butter

The fatty acid composition of 11 brands of peanut butter and paste freshly prepared from roasted peanuts was analyzed with emphasis on isomeric trans-fatty acids. No trans-fatty acids were detected in any of the samples in an analytical system with a detection threshold of 0.01% of the sample weight. Hydrogenated vegetable oils are added to peanut butters at levels of 1--2% to prevent oil separation. Some hydrogenated vegetable oils are known to be sources of trans-fatty acids in the human diet. The addition of these products was not found to result in measurable amounts of trans-fatty acids in the peanut butters analyzed.     

Simple, rapid cleanup method for analysis of aflatoxins and comparison with various methods

A method is described for simple and rapid determination of aflatoxins in corn, buckwheat, peanuts, and cheese. Aflatoxins were extracted with chloroform-water and were purified by a Florisil column chromatographic procedure. Column eluates were concentrated and spotted on a high performance thin layer chromatographic (HPTLC) plate, which was then developed in chloroform-acetone (9 + 1) and/or ether-methanol-water (94 + 4.5 + 1.5) or chloroform-isopropanol-acetone (85 + 5 + 10). Each aflatoxin was quantitated by densitometry. The minimum detectable aflatoxin concentrations (micrograms/kg) in various test materials were 0.2, B1; 0.1, B2; 0.2, G1; 0.1, G2; and 0.1, M1. Recoveries of the aflatoxins added to corn, peanut, and cheese samples at 10-30 micrograms/kg were greater than 69% (aflatoxin G2) and averaged 91%, B1; 89%, B2; 91%, G1; 78%, G2; and 92%, M1. The simple method described was compared with the AOAC CB method, AOAC BF method, and AOAC milk and cheese method. These methods were applied to corn, peanut, and cheese composites spiked with known amounts of aflatoxins, and to naturally contaminated buckwheat and cheese. Recoveries were much lower for the BF method compared with our simple method and the CB method.     

Rapid determination of aflatoxins in raw peanuts by liquid chromatography with postcolumn iodination and modified minicolumn cleanup

A method is described for rapid cleanup followed by reverse-phase liquid chromatographic (LC) quantitation of aflatoxins in raw peanuts. A modified minicolumn cleanup is used for sample preparation, and a preliminary estimation of aflatoxin content by minicolumn can be made so that highly contaminated samples can be diluted before LC analysis. The use of the simple, quick minicolumn cleanup eliminates the need for further column or cartridge cleanup, thus greatly reducing sample preparation time. Sensitive quantitation is achieved using a phenyl column, a mobile phase of water-tetrahydrofuran (80 + 20, v/v), and postcolumn derivatization with water-saturated iodine followed by fluorescence detection. The recoveries of aflatoxins B1, B2, G1, and G2 from peanut meal spiked at 3 levels ranged from 71.7 to 88.3% (average 80%) with coefficients of variation from 2.7 to 10.4%.     

Evaluation of APHA and AOAC II methods for phosphatase in butter and differentiation of milk and microbial phosphatases by agarose-gel electrophoresis

Salted and unsalted butters with 3 levels of phosphatase were prepared with both raw and pasteurized cream containing 36% fat. Test samples were analyzed for phosphatase by the modified method of the American Public Health Association (APHA) and the official AOAC method, 16.256 (1984, 14th Ed., 1990 15th Ed., 946.02). In the APHA method, weighing of solid frozen butter for testing yielded repeatable results. Addition of 0.0-1.0 mg magnesium to the butter had little effect on phosphatase activity in the APHA modified rapid colorimetric method (MRCM), but caused the phosphatase activity to decrease in the AOAC method. Phosphatase in salted and unsalted butters was quite stable at -17 +/- 1 degrees C and at 3.0 +/- 0.5 degrees C; however, within 2 to 4 days, freshly prepared butters stored at 22 +/- 1 degrees C developed reactivated and/or microbial phosphatases that were both heat-labile and heat-stable. At 22 +/- 1 degrees C, frozen butters showed decreased milk phosphatase activity before producing microbial phosphatase. Heat-labile phosphatases in salted and unsalted butters were inactivated at 62.8 degrees C for 10 min, and the phosphatase lability was partially due to the heat-denaturing effect of NaCl in salted butter. Some heat-stable phosphatases in unsalted butter survived at 66 degrees C for 30 min. Differentiation of milk phosphatase from microbial phosphatases was difficult by both methods; however, they were successfully differentiated by the agarose-gel electrophoretic technique.     

Evaluation of enzyme-linked immunosorbent assay of cleanup for thin-layer chromatography of aflatoxin B1 in corn, peanuts, and peanut butter

A simple, rapid enzyme-linked immunoassay (ELISA) was used to evaluate the performance of each step (extraction, filtration, solvent partition, and silica gel column chromatography) of a solvent-efficient thin-layer chromatographic (TLC) method which is undergoing interlaboratory collaborative study for the determination of aflatoxin B1 in corn, raw peanuts, and peanut butter. The apparent average recoveries using the ELISA method were about 30 to 50% higher than those using the TLC method if only the amount of B1 added to the samples was used in the calculations. After the cross-reaction of the antibody with other aflatoxins added to the samples was considered, the amounts recovered approached the levels of aflatoxins added in all 3 commodities tested. With no cleanup treatment, ELISA recoveries at aflatoxin B1 levels above 7.5 ng/g were 84, 79, and 103% for corn, raw peanuts, and peanut butter, respectively. The coefficients of variation were between 5.2 and 25.2%. With each cleanup step in the TLC method, ELISA detected a progressive decrease in recovery from 150.5 to 105.3% (before correction for the presence of other aflatoxins) or from 93.5 to 65.4% (after correction for other aflatoxins) of B1 added to the samples. The ELISA data support the conclusion obtained from previous studies that cleanup treatments were not necessary in the ELISA. When large amounts of other aflatoxins are present, an understanding of the cross-reactivity of antibody with other aflatoxins in the ELISA is essential for final interpretation of the data.     

Liquid chromatographic method for determination of aflatoxins B1, B2, G1, and G2 in corn and peanut products: collaborative study

A collaborative study of a liquid chromatographic method for the determination of aflatoxins B1, B2, G1, and G2 was conducted in laboratories located in the United States, Canada, South Africa, and Switzerland. Twenty-one artificially contaminated raw peanuts, peanut butter, and corn samples containing varying amounts of aflatoxins B1, B2, G1, and G2 were distributed to participating laboratories. The test portion was extracted with methanol-0.1N HCl (4 + 1), filtered, defatted with hexane, and then partitioned with methylene chloride. The concentrated extract was passed through a silica gel column. Aflatoxins B1 and G1 were derivatized with trifluoroacetic acid, and the individual aflatoxins were determined by reverse-phase liquid chromatography with fluorescence detection. Statistical analysis of the data was performed to determine or confirm outliers, and to compute repeatability and reproducibility of the method. For corn, relative standard deviations for repeatability (RSDr) for aflatoxin B1 ranged from 27.2 to 8.3% for contamination levels from 5 through 50 ng/g. For raw peanuts and peanut butter, RSDr values for aflatoxin B1 were 35.0 to 41.2% and 11.2 to 19.1%, respectively, for contamination levels from 5 through 25 ng/g. RSDr values for aflatoxins B2, G1, and G2 were similar. Relative standard deviations for reproducibility (RSDr) for aflatoxin B1 ranged from 15.8 to 38.4%, 24.4 to 33.4%, and 43.9 to 54.0% for corn, peanut butter, and raw peanuts, respectively. The method has been adopted official first action for the determination of aflatoxins B1, B2, G1, and G2 in peanut butter and corn at concentrations greater than or equal to 13 ng total aflatoxins/g.     

A validated multianalyte LC-MS/MS method for quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples

This study was designed to develop a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples with particular focus on the optimization of the sample preparation protocol and method validation. All 25 mycotoxins were extracted in a single step with a mixture of methanol/ethyl acetate/water (70:20:10, v/v/v). The method limits of quantification (LOQ) varied from 0.3 μg/kg to 106 μg/kg. Good precision and linearity were observed for most of the mycotoxins. The method was applied for the analysis of naturally contaminated peanut cake, cassava flour and maize samples from the Republic of Benin. All samples analyzed (fifteen peanut cakes, four maize flour and four cassava flour samples) tested positive for one or more mycotoxins. Aflatoxins (total aflatoxins; 10-346 μg/kg) and ochratoxin A (相似文献   

Improved enzyme-linked immunosorbent assay for aflatoxin B1 in agricultural commodities

An improved enzyme-linked immunosorbent assay (ELISA) for aflatoxin B1 in cornmeal and peanut butter was developed. Aflatoxin B1 in cornmeal and peanut butter samples was extracted with 70% methanol in water containing 1% dimethylformamide diluted with assay buffer to a final concentration of 7.0% methanol, and directly subjected to an ELISA procedure that took less than 1 h for quantitative analysis and less than 30 min for screening tests. Analytical recoveries for 5-100 ppb B1 added to the cornmeal and peanut butter were 91 and 95.4%, respectively. The interwell and interassay coefficient of variation was 10% or less at the 20 ppb level and above. Agreement for B1 levels in more than 30 naturally contaminated corn, mixed feed, and peanut butter samples was excellent between the ELISA data and the data obtained from different independent laboratories using TLC or other analytical methods.     

Reverse phase liquid chromatographic determination and confirmation of aflatoxin M1 in cheese

A systematic method is proposed for determination and confirmation of aflatoxin M1 in cheese by liquid chromatography (LC). A sample of cheese is extracted with chloroform, cleaned up on 2 silica gel columns followed by a Sep-Pak C18 cartridge, and chromatographed on a 5 microns octadecyl silica column with fluorometric detection. The sample extract or standard is treated with n-hexane-trifluoroacetic acid (TFA) (4 + 1) for 30 min at 40 degrees C. Analysis by LC with TFA-treatment of the extract provides quantitative data. Multiple assays of 5 samples of Gouda cheese spiked with aflatoxin M1 at levels of 0.5, 0.1, and 0.05 ng/g showed average recoveries of 93.2, 91.6, and 92.4%, with coefficients of variation of 2.63, 3.97, and 4.52%, respectively. Assay of 5 naturally contaminated cheeses resulted in 0.051-0.448 ng/g of aflatoxin M1. Limit of quantitation is about 0.01 ng/g. The identity of aflatoxin M1 is confirmed by treating aflatoxin M1 or the M2a derivative with TFA-methanol (or ethanol) (3 + 1). The TFA-methanol reaction products of M2a could be detected quantitatively.     

Analysis of the Fusarium mycotoxins fusaproliferin and trichothecenes in grains using gas chromatography-mass spectrometry

A method is described using gas chromatography-mass spectrometry (GC-MS) for the simultaneous detection of the Fusarium mycotoxins fusaproliferin and seven trichothecenes from grains. Sample purification of the raw extract was carried out with commercial solid phase extraction columns, and the recovery of the more polar analytes was increased by rinsing the column with acetonitrile. A significant matrix effect was found for the analysis of fusaproliferin and trichothecenes; thus, the calibrants should be prepared in a blank matrix. The response was linear in the range used. The mean recovery for fusaproliferin was 60.4 or 62.9%, depending on the spiking level. With respect to the trichothecenes, the recovery was generally higher (70.2-125.3%). The method proved to be repeatable for the analysis of fusaproliferin and trichothecenes. The limit of detection for fusaproliferin in the blank matrix mixture was 50 microg/kg, and that for trichothecenes was 5-15 microg/kg. Thirty-eight Finnish grain samples were analyzed for fusaproliferin and trichothecenes with the method developed. Fusaproliferin was not detected in any of the samples. The mean levels of deoxynivalenol, 3-acetyldeoxynivalenol, nivalenol, HT-2 toxin, and T-2 toxin in Finnish grain samples were 272, 17, 150, 40, and <20 microg/kg, respectively.     

用于污染黄曲霉毒素花生分选的荧光信号研究

为在加工前将黄曲霉毒素超限的带衣花生米从原料中剔除,参照已有的色选系统,提出一种依据黄曲霉毒素含量超限带衣花生米的专属荧光信号进行逐粒分选的技术构想。采用Cary Eclipse荧光分光光度计测定100粒外观具有代表性的带衣花生米表面的紫外-荧光规律,通过与免疫亲和层析净化荧光光度法(GB/T18979-2003)检测结果对比,判定了黄曲霉毒素超限带衣花生米的荧光光谱特征;通过绘制450/490、460/490荧光强度比值的箱线图,评估了表面荧光法判断黄曲霉毒素超限带衣花生米的准确率;在搭建的荧光成像系统上,对黄曲霉毒素超限带衣花生米进行了荧光成像。检测发现,在365 nm波长激发下,黄曲霉毒素超限带衣花生米在420~460 nm处有荧光峰;以450/490荧光强度比值为依据剔除超限值带衣花生米的判断准确率为81%;a.u.40的带衣花生米可在图像中呈现亮蓝荧光光斑。表明表面荧光信号可作为带衣花生米在线、无损、逐粒分选的专属光学信号,用于黄曲霉毒素超限带衣花生米的剔除。     

基于电子鼻的花生有害霉菌种类识别及侵染程度定量检测

针对花生霉变传统分析方法操作繁琐、时效性差等不足,该研究拟利用电子鼻气体传感技术建立起花生有害霉菌污染的快速检测方法。辐射灭菌花生籽粒分别接种5种谷物中常见有害霉菌(黄曲霉3.17、黄曲霉3.395 0、寄生曲霉3.395、寄生曲霉3.012 4和赭曲霉3.648 6),并于26℃、80%相对湿度条件下储藏9 d至严重霉变。利用电子鼻气体传感器获取不同储藏时期(0、3、6、9 d)花生样品的整体挥发性气味信息。最后,结合多元统计分析方法对电子鼻传感器响应信号进行特征提取,建立了花生中有害霉菌污染程度的定性定量分析模型。结果显示,主成分分析法(principal component analysis,PCA)可成功区分不同霉菌侵染程度的花生样品,线性判别分析(linear discriminant analysis,LDA)模型对样品不同储藏天数判别的准确率均达到或接近100%。花生中菌落总数的偏最小二乘回归分析(partial least squares regression,PLSR)模型的预测决定系数和预测相对均方根误差分别达到0.814 5和0.244 0 lg(CFU/g)。结果表明,应用电子鼻技术快速检测储藏期间花生霉变状况具有一定可行性,可为利用气味信息实现粮食霉菌污染的在线监测提供理论参考。     

Thin layer chromatography and densitometric determination of aflatoxins in mixed feeds containing citrus pulp

A method for the accurate one-dimensional thin layer chromatographic (TLC) determination of aflatoxins B1, B2, G1, and G2 in mixed feeds is presented. The aflatoxins are extracted from the sample with chloroform and purified by solvent partitioning. Each aflatoxin is separated from pulp interference by thin layer chromatography on aluminum-backed silica plates. The separated aflatoxins are detected by fluorescence densitometry. Average recoveries for samples spiked from 10 to 100 ppb B1 and G1 and from 3 to 30 ppb B2 and G2 are 82, 84, 95, and 94% for B1, B2, G1, and G2, respectively. The above recovery data, when analyzed for overall method repeatability, produced relative standard deviations of 6.8, 4.3, 6.9, and 7.6% for B1, B2, G1, and G2, respectively. Minimum detection level is less than 1 ppb for each aflatoxin. B1 is confirmed by trifluoroacetic acid derivative formation on a silica TLC plate.     

Survey of aflatoxins, ochratoxin A, zearalenone, and sterigmatocystin in some Brazilian foods by using multi-toxin thin-layer chromatographic method

A previously published method for ochratoxin A was evaluated and proved appropriate for simultaneous determination of aflatoxins, ochratoxin A, sterigmatocystin, and zearalenone, with considerable savings in time and reagent costs. The detection limits were 2, 5, 15, and 55 micrograms/kg, respectively. The recoveries and coefficients of variation obtained with artificially contaminated samples were 91-101% and 0-16% for aflatoxin B1, 98-117% and 0-17% for sterigmatocystin, and 96-107% and 0-17% for zearalenone, respectively. The coefficients of variation for naturally contaminated samples (aflatoxins in rice and ochratoxin A in beans) ranged from 0 to 8%. The method was used to survey 296 samples that included 10 cultivars of dried beans, 8 types of corn products, 3 types of cassava flour, and both polished and parboiled rice between May 1985 and June 1986 in Campinas, Brazil. Only aflatoxin B1 (9 samples, 20-52 micrograms/kg), aflatoxin G1 (4 samples, 18-31 micrograms/kg), and ochratoxin A (5 samples, 32-160 micrograms/kg) were found. The average contamination percentage was 4.7%; beans showed the highest (6.6%) and rice showed the lowest (3.3%) incidence rates. Zearalenone and sterigmatocystin were not detected. Positive samples were confirmed by chemical derivatization, corroborated by development in 3 solvent systems.     

我国花生土壤黄曲霉菌分布与产后花生黄曲霉毒素污染相关性研究

为了解我国花生土壤黄曲霉分布及产毒特征与产后花生黄曲霉毒素污染相关性,本试验从我国4个典型花生产区黄河流域产区(河北保定)、西北产区(新疆吐鲁番)、长江流域产区(湖北黄冈、四川南充)和东南沿海产区(广东湛江)采集花生土壤样品124份。通过对我国不同产区花生土壤中黄曲霉菌的分布及产毒特征研究,评估我国不同产区花生黄曲霉毒素污染风险。结果表明,湖北黄冈和四川南充土壤中黄曲霉检出率、带菌量和黄曲霉毒素B₁(AFB₁)量均高于其他地区,产后花生受AFB₁污染风险最高,其次为广东湛江和新疆吐鲁番,河北保定花生受AFB₁污染风险最低。从上述4个产区收集64份花生样品开展产后花生AFB₁污染调查,发现湖北黄冈花生AFB₁污染情况最严重,检出率为57%,超标率为10%,其次为四川南充和广东湛江,新疆吐鲁番和河北保定花生受AFB₁污染较轻。风险评估结果与实际检测结果一致,表明花生土壤中黄曲霉菌数量、产毒菌比例及产毒能力与产后花生AFB₁污染呈正相关性。本研究结果为花生黄曲霉毒素污染预警及综合防控提供了理论依据和数据支撑。     

Determination of 49 organophosphorus pesticide residues and their metabolites in fish, egg, and milk by dual gas chromatography-dual pulse flame photometric detection with gel permeation chromatography cleanup

A new method for the quantitative determination of 49 kinds of organophosphorus pesticide residues and their metabolites in fish, egg, and milk by dual gas chromatography-dual pulse flame photometric detection was developed. Homogenized samples were extracted with acetone and methylene chloride (1 + 1, v/v), and then the extracts were cleaned up by gel permeation chromatography (GPC). The response of each organophosphorus pesticide showed a good linearity with its concentration; the linearity correlation was not less than 0.99. The detection limits (S/N = 3) of pesticides were in the range of 0.001-0.025 mg kg?1. The recovery experiments were performed by blank sample spiked at low, medium, and high fortification levels. The recoveries for fish, egg, and milk were 50.9-142.2, 53.3-137.2, and 50.3-139.4% with relative standard deviations (RSD, n = 6) of 2.3-24.9, 4.3-26.7, and 2.8-32.2%, respectively. The method was applied to detect organophosphorus pesticides in samples collected from the market, and satisfactory results were obtained. This quantitative method was highly sensitive and exact and could be applied to the accurate determination of organophosphorus contaminants in fish, egg, and milk.     

Gas chromatographic determination of deoxynivalenol in wheat with electron capture detection: collaborative study

Ten laboratories participated in a collaborative study of a method for the determination of deoxynivalenol in wheat by gas chromatography with electron capture detection. Each laboratory analyzed 6 samples in duplicate. Each collaborator received samples spiked at the 100.3, 501.3, and 1002.6 ng/g levels; a control sample; and 2 naturally contaminated samples. The average recovery (outliers excluded) for the spiked samples was 92.2%. The mean repeatability and reproducibility, respectively, were 32.2 and 41.3% for the spiked samples and 30.9 and 47.6% for the naturally contaminated samples. The method was adopted official first action.