Liquid chromatographic determination of aflatoxin levels in peanut butters using an immunoaffinity column cleanup method: international collaborative trial

Thirteen laboratories in 7 different countries participated in a collaborative trial to evaluate the immunoaffinity column cleanup procedure with quantitation by fluorescence liquid chromatography (post-column derivatization) for the determination of aflatoxins in peanut butters. Participants were sent 10 randomly numbered samples of roasted peanut butter for analysis (5 pairs of undisclosed duplicates). Two of the pairs were "blank" peanut butters to which aflatoxin standards had been added; these "spiked" samples were used for recovery purposes. The other 3 pairs of samples were a nominal "blank" and 2 naturally contaminated peanut butters. A full statistical presentation of the results is given. Coefficients of variation (CVs) for the total aflatoxin determinations for mean levels of 4, 15, and 38 microns/kg were between 32 and 44% for the blank and 2 trial samples. Recovery levels for the 2 spiked samples were 51-67%, with aflatoxin B1 recovery of 60%. Relative standard deviations for method repeatability (RSDr) and reproductibility (RSDR) for the 3 trial samples were 15-26% and 33-45%, respectively.     

Collaborative study of a method for chemical confirmation of the identity of aflatoxin.

The chemical method for confirmation of the identity of aflatoxin by derivative formation directly on the TLC plate was studied collaboratively by 8 participants. The results show that aflatoxin B-1 was confirmed in 17 of 17 sample extracts representing 15 mu-g aflatoxin B-1/kg peanut butter, in 13 of 16 extracts representing 5 mu-g/kg, and in none of the 7 aflatoxin-free extracts. Collaborators commented that the method was easily performed and gave good results. The method has been adopted as official first action.     

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.     

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.     

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.     

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.     

Value assignment of nutrient and aflatoxin concentrations in standard reference material 2387 peanut butter

Standard Reference Material (SRM) 2387 peanut butter was recently issued, and the process used for value assignment of nutrient and aflatoxin concentrations is reported herein. Values were assigned using data provided by the National Institute of Standards and Technology (NIST) and collaborating laboratories. SRM 2387 is intended for use as a primary material for assigning values to in-house control materials and for validation of analytical methods for measurements in peanut butter and similar high-fat matrixes. SRM 2387 lies in sector 3 of AOAC International's fat-protein-carbohydrate triangle. With the addition of SRM 2387, NIST now offers materials within-or on the borders between-all sectors of the triangle. The Certificate of Analysis for SRM 2387 provides assigned values for concentrations of fatty acids, proximates, elements, and total dietary fiber, for which product labeling is required by the Nutrition Labeling and Education Act of 1990, as well as several vitamins, amino acids, and aflatoxins, for which labeling is not required. (Aflatoxin levels in peanut butter are regulated by the Food and Drug Administration.)     

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.     

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   

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.     

Development of milk powder reference materials certified for aflatoxin M1 content (Part II): Certification of milk powder RM 283

The development of a full cream milk powder reference material, certified for its aflatoxin M1 content (target concentration: 0.1 microgram/kg), is described. The material (RM 283) was prepared and certified within the Reference Material Programme of the Community Bureau of Reference, along with other members of a series of milk powder reference materials. Homogeneity, evaluated by determining the aflatoxin M1 content of 30 units, was found to be acceptable (coefficient of variation of analysis results: 9.1%); stability has been demonstrated in a long-term study. The certification exercise involved 7 laboratories. Calibration, control of recoveries, blank values, and independence of the replicate measurements were emphasized. All sets of results of the certification exercise were accepted for statistical evaluation. A certified value for the aflatoxin M1 content: 0.09(+0.04)(-0.02) micrograms/kg was derived. The certification of RM 283 completes the series of 4 milk powder reference materials having certified aflatoxin M1 contents.     

Visual and semiquantitative spectrophotometric ELISA screening method for aflatoxin B1 in corn and peanut products: follow-up collaborative study

A joint AOAC/IUPAC (International Union of Pure and Applied Chemistry) interlaboratory study of an enzyme-linked immunosorbent screening assay (ELISA) for aflatoxins was conducted in laboratories in Canada, France, Japan, The Netherlands, Switzerland, Tunisia, and the United States. Twelve raw and roasted peanut and corn portions containing various concentrations of natural aflatoxins and supplemented when appropriate with aflatoxin B1 were distributed to participating laboratories for testing. The assay is based on competition between an enzyme-conjugated aflatoxin B1 and (free) aflatoxins in the test sample for aflatoxin-specific antibodies coated onto interior surfaces of microtiter wells. After a wash step to remove all unbound aflatoxins, a substrate added to each well is catalyzed from a colorless to a blue solution by any bound enzyme-conjugated aflatoxin B1 present. The intensity of the color decreases as the amount of free aflatoxin B1 in the test portion increases. Final determination of aflatoxin concentrations can be made by either visual comparison with standard solutions or spectrophotometric comparisons (at 650 nm) to knowns. Overall correlation was good between ELISA and thin-layer chromatographic results for corn and roasted peanut products, with 93 and 98% correct responses for visual and instrumental determinations, respectively. For instrumental determinations of aflatoxin in corn and roasted peanuts in the less than or equal to 20 ng/g range, the relative standard deviations for repeatability (RSDr) were 14.9 and 41.4%, respectively, and the relative standard deviations for reproducibility (RSDR) were 45.7 and 43.5%, respectively. For instrumental determination of greater than 20 ng/g, the respective RSDr and RSDR values were 19.4 and 52.7% for corn and 23.3 and 23.3% for roasted peanuts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Collaborative study of a screening method for the detection of aflatoxins in mixed feeds, other agricultural products, and foods.

A screening method for aflatoxins was collaboratively tested on 11 different agricultural and food products: white and yellow corn, peanuts, peanut butter, pistachio nuts, peanut meal, cottonseed meal, chicken, pig, and turkey starter rations, and dairy cattle feed. The method involves a rapid extraction and cleanup procedure followed by the detection of total aflatoxins (B1 + B2 + G1 + G2) as a fluorescent band on the Florisil layer of a Velasco-type minicolumn. The results of 32 collaborators from 10 different countries are presented. Samples containing 0, 5, 10, 15, 20, and 25 mug aflatoxins/kg were analyzed. Eighty-four per cent of the negative samples and 89% of the samples containing 10-25 mug total aflatoxins/kg were correctly identified. This method has been adopted as official first action for the detection of aflatoxins in corn, peanuts, peanut butter, peanut meal, cottonseed meal, mixed feeds, and pistachio nuts.     

Caffeine as main interfering compound in radioimmunoassay of aflatoxin B1 in coffee samples

The content of caffeine in coffee extracts prepared for radioimmunoassay of aflatoxin B1 was determined by gas chromatography. The extracts from coffee beans and decaffeinated coffee contained 1.76-4.60 and 0.71-0.85 g caffeine/kg, respectively. These concentrations of caffeine caused false results in radioimmunoassay of aflatoxin B1 in the range 1.0-2.8 micrograms/kg for coffee beans and 0.3-0.4 micrograms/kg for decaffeinated coffee.     

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。     

Simultaneous thin layer chromatographic determination of aflatoxin B1 and ochratoxin A in black olives

A screening method has been developed for simultaneous determination of aflatoxin B1 and ochratoxin A in black olives. The technique includes extraction of both mycotoxins with aqueous methanol, cleanup using lead acetate, defatting with hexane, partitioning in chloroform, and thin layer chromatography. Detection limits are 5-7 micrograms aflatoxin B1 and 20 micrograms ochratoxin A/kg.     

Rapid quantitation and confirmation of aflatoxins in corn and peanut butter, using a disposable silica gel column, thin layer chromatography, and gas chromatography/mass spectrometry

A simple, rapid, and solvent-efficient method for determining aflatoxins in corn and peanut butter is described. Aflatoxins B1, B2, G1, and G2 were extracted from 50 g sample with 200 mL methanol-water (85 + 15). A portion of the extract was diluted with 10% NaCl solution to a final concentration of 50% methanol, and then defatted with hexane. The aflatoxins were partitioned into chloroform. The chloroform solution was evaporated, and the residue was placed on a 0.5 g disposable silica gel column. The column was washed with 3 mL each of hexane, ethyl ether, and methylene chloride. Aflatoxins were eluted with 6 mL chloroform-acetone (9 + 1). The solvent was removed by evaporation on a steam bath, and the aflatoxins were determined using thin layer chromatography (TLC) with silica gel plates and a chloroform-acetone (9 + 1) developing solvent. Overall average recovery of aflatoxin B1 from corn was 82%, and the limit of determination was 2 ng/g. For mass spectrometric (MS) confirmation, aflatoxin B1 in the extract from 3 g sample (20 ng/g) was purified by TLC and applied by direct on-column injection at 40 degrees C into a 6 m fused silica capillary gas chromatographic column. The column was connected directly to the ion source. After injection, the temperature was rapidly raised to 250 degrees C, and the purified extract was analyzed by negative ion chemical ionization MS.     

Thin layer chromatographic determination of aflatoxins, ochratoxins, sterigmatocystin, zearalenone, citrinin, T-2 toxin, diacetoxyscirpenol, penicillic acid, patulin, and penitrem A.

A general method is described for determining 16 mycotoxins in mixed feeds and other food products used in the manufacture of these feedstuffs. The mycotoxins are extracted and cleaned up by extracting with solvents of different pH. Thin layer chromatography is used to separate the toxins; toxins are then quantitated by the limit detection method. The minimum detectable concentration of mycotoxins in various products is: aflatoxin B1 or G1, 4--5 micrograms/kg; ochratoxin A or ethyl ester A 140--145 micrograms/kg; citrinin 600--750 micrograms/kg; zearalenone, 410--500 micrograms/kg; sterigmatocystin, 140--145 micrograms/kg; diacetoxyscirpenol, 2400--2600 micrograms/kg; T-2 toxin, 800--950 micrograms/kg; patulin, 750--800 micrograms/kg; penitrem A 14,000--14,500 micrograms/kg; penicillic acid 3400--3650 micrograms/kg.     

Determination of aflatoxins in vegetable oils

A simple method is proposed for determination of aflatoxins in vegetable oils. The method was successfully applied to both crude and degummed oils. The oil sample, dissolved in hexane, was applied to a silica column and washed with ether, toluene, and chloroform; aflatoxins were eluted from the column with chloroform-methanol (97 + 3). As quantitated by thin layer chromatography and liquid chromatography, the oils analyzed contained aflatoxin B1 at levels of 5-200 micrograms/kg. Recoveries of aflatoxin B1 standards added to aflatoxin-free oils were between 89.5 and 93.5%, with coefficients of variation of 6.3-8.0%.     

Development of milk powder reference materials certified for aflatoxin M1 content (Part I)

The development of 3 full-cream milk powder reference materials, certified for their aflatoxin M1 content, is described. The materials were prepared and certified within the Reference Material Programme of the Community Bureau of Reference (BCR). The 3 reference materials, RMs 282, 284, and 285, contain aflatoxin M1 at concentrations of less than 0.05, 0.31 +/- 0.06, and 0.76 +/- 0.05 micrograms/kg, respectively. The preparation, testing for homogeneity, stability of the reference materials, and the certification exercise, which was preceded by 2 intercomparisons of methods, are discussed. Particular emphasis was placed on the independence of the measurements in the certification exercise and the control of errors associated with extraction efficiency and the aflatoxin M1 calibrant. Finally, some guidance is given on avoiding the principal sources of errors in the determination of aflatoxin M1. Details concerning the supply of the reference materials will be provided by BCR on request.