Distribution of aflatoxin in pistachios. 6. Seller's and buyer's risk.

The seller's risk-the probability of a set of samples exceeding an agreed upon aflatoxin level when the lot mean does not-and the buyer's risk-the probability of a lot exceeding this level when a set of samples do not-have been computed using a parametrized experimental aflatoxin distribution and Monte Carlo simulation. The calculations are exemplified using the proposed EC standards (three 10 kg samples, 4 ng/g of total aflatoxin, basis kernels only) as well as for samples up to 250 kg and for varied lot aflatoxin levels. It is found that within this sample size range the seller's risk is as high as 42% at 10 kg and increases with increasing sample size to 80% at 250 kg. Only by reducing lot levels to 0.2 ng/g of total aflatoxin, basis kernels, can the risk be brought down to 2.5%, independent of sample size. The buyer's risk is as high as 58% at 10 kg but falls to 11% at 250 kg samples. The implications for both seller and buyer strategies are discussed.     

Effect of pressure cooking on aflatoxin B1 in rice

The effect of pressure cooking on aflatoxin residues in polished rice was conducted to determine reduction of aflatoxin and mutagenic potentials. Three rice lots consisting of naturally contaminated, A. parasiticus-infested, and aflatoxin-spiked rice were steamed by ordinary and pressure cookers after they were washed with water. They were chemically analyzed for aflatoxins using a silica solid phase extraction tube and high-performance liquid chromatography (HPLC)-fluorescence detection (FD), and the presence of aflatoxin residues was confirmed using HPLC-electrospray ionization (ESI)-mass spectrometry (MS). An in vitro mutagenicity test with Salmonella typhimurium TA100 was employed to verify the results based on chemical analyses. The aflatoxin loss (78-88%) was notable after pressure cooking, and the reduction of aflatoxin-induced mutagenic potential (68-78%) was in good agreement with the HPLC results. It can be concluded that Koreans are safe from the aflatoxin-related risk if a pressure cooker is employed for cooking rice. The average Korean daily intake of aflatoxin through the consumption of staple rice would fall to 0.15 ng/kg bw/day, which would not exceed the established tolerable daily intake (0.40 ng/kg bw/day).     

Dependence of aflatoxin in almonds on the type and amount of insect damage

The aflatoxin distribution of single insect damaged Nonpareil almonds (1999 crop) has been measured. Separate distributions were obtained for pinhole, insect (feeding), and gross damage. Only a low level of aflatoxin contamination ( = 0.0003 ng/g) was found for pinhole-only damaged nuts. The distributions for insect and gross damage did not differ, but did differ significantly from the distribution previously obtained for gross damaged Ne Plus almonds from a different producer (Schatzki, T. F.; Ong, M. S. J. Agric. Food Chem. 2000, 48, 489-492; also 1999 crop). The Nonpareil almond distribution could be explained on the basis of a preharvest hull splitting, similar to previous results in pistachios (0-4 weeks versus 2-6 weeks preharvest). The Ne Plus distribution differs in detail from pistachio results and from the Nonpareil results found here. This may indicate additional cultural damage of Ne Plus almonds around harvest time and/or use of different sorting parameters. Aflatoxin lot averages of 31.7 and 3.47 ng/g were obtained for 100% insect damaged Ne Plus and Nonpareil almonds, respectively. (The previous Ne Plus work contained a calculation error, which is corrected here.) The distribution functions were used to compute the seller's risk of nonacceptance of lots in the European Union. To obtain a 95% acceptance rate, aflatoxin B(1) levels of 0.12 and 0.22 ng/g would be required, which would correspond to 3.8 and 1.2% (feeding and gross) insect damage in Nonpareil and Ne Plus almond lots, respectively.     

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   

Rapid, non-destructive selection of peanuts for high aflatoxin content by soaking and tandem mass spectrometry

Peanut lots are subject to aflatoxin levels high enough to cause concern to health agencies and trade channels. A possible solution would be to mechanically sort out high aflatoxin nuts from the process stream. Only highly contaminated nuts would need to be removed. However, there exists at present no sorting mechanism which meets commercial needs of adequate reduction and product preservation. To build such a sorter requires knowledge of the properties that can be used for sorting. The first step in the design is to select on the order of one hundred undamaged contaminated nuts which can be compared with noncontaminated ones. Because contaminated nuts are rare, a very large number of nuts needs to be examined nondestructively. We present a method to rapidly carry out such a selection. The method is based on dipping nuts into extraction fluid and examining the resulting fluid by tandem MS without preliminary cleanup. This method has been applied to examine over 65,000 nuts, yielding approximately 120 nuts, each containing more than 250-43000 ng/g aflatoxin (depending on process stream).     

Evaluation of a testing program for aflatoxin in corn

A computer model that accounts for sampling and analytical variability was developed to simulate the aflatoxin testing program administered by the North Carolina Department of Agriculture (NCDA) to regulate aflatoxin in corn meal. Monte Carlo solution techniques were employed to account for conditional probabilities that rise from multiple samples being used in the testing program. The NCDA testing program was then evaluated by applying the computer model to a hypothetical group of 1000 corn meal lots with the same distribution of aflatoxin concentrations as was observed among aflatoxin assays made by NCDA on commercial lots of corn meal from 1977 to 1980. The average of the 1000 lots assayed was 17.7 parts per billion (ppb). The model predicted that 79.5% of the lots would be accepted and 20.5% of the lots would be rejected by the NCDA testing program. The accepted and rejected lots contained an average of 5.7 and 64.2 ppb aflatoxin, respectively. The testing program accepted 7.3% of the lots with more than 20 ppb aflatoxin (consumers' risk) and rejected 1.0% of the lots with 20 ppb or less (processors' risk). A correct decision was made 94% of the time.     

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.     

Thin layer chromatographic determination of aflatoxin in corn dust

Methods adopted by the AOAC and the American Association of Cereal Chemists for determining aflatoxin in corn were modified, and techniques were developed for application to samples of less than 1 to 10 g instead of the specified 50 g samples. Analysis included chloroform extraction of dust samples or dust collected from glass fiber filters, purification of extracts on a silica gel column of appropriate size, and measurement of aflatoxin by either 1- or 2-dimensional thin layer chromatography (TLC). The solvent for 1-dimensional TLC was chloroform-acetone-water (91 + 9 + 1). Solvents for 2-dimensional TLC were, first direction, ether-methanol-water (95 + 4 + 1, lined tank) and second direction, chloroform-acetone-water (91 + 9 + 1, unlined tank), or first direction, chloroform-acetone-water (91 + 9 + 1, unlined tank) and second direction, toluene-ethyl acetate-formic acid (60 + 30 + 10, unlined tank). When samples weighed less than or equal to 0.1 g, the entire concentrated extract was applied to the TLC plate. About 0.5-1.0 ng aflatoxin B1 could be detected on the plate, making the limit of detection about 9 ng/g for 0.1 g samples.     

Use of ten gram samples of corn for determination of mycotoxins

Data were gathered, during a study on the development of an automated system for the extraction, cleanup, and quantitation of mycotoxins in corn, to determine if it was scientifically sound to reduce the analytical sample size. Five, 10, and 25 g test portions were analyzed and statistically compared with 50 g test portions of the same composites for aflatoxin concentration variance. Statistical tests used to determine whether the 10 and 50 g sample sizes differed significantly showed a satisfactory observed variance ratio (Fobs) of 2.03 for computations of pooled standard deviations; paired t-test values of 0.952, 1.43, and 0.224 were computed for each of the 3 study samples. The results meet acceptable limits, since each sample's t-test result is less than the published value of the /t/, which is 1.6909 for the test conditions. The null hypothesis is retained since the sample sizes do not give significantly different values for the mean analyte concentration. The percent coefficients of variation (CVs) for all samples tested were within the expected range. In addition, the variance due to sample mixing was evaluated using radioisotope-labeled materials, yielding an acceptable CV of 22.2%. The variance due to the assay procedure was also evaluated and showed an aflatoxin B, recovery of 78.9% and a CV of 11.4%. Results support the original premise that a sufficiently ground and blended sample would produce an analyte variance for a 10 g sample that was statistically comparable with that for a 50 g sample.     

Simulation of aflatoxin testing plans for shelled peanuts in the United States and in the export market

The 1987 United States aflatoxin testing plan for shelled peanuts was designed with a final accept level of 25 parts per billion (ppb) total aflatoxin. Some of the importers of U.S. peanuts use aflatoxin testing plans with accept levels lower than 25 ppb. For example, the accept level of a testing plan used in The Netherlands is 5 ppb B1 or 10 ppb total aflatoxin. Whenever export lots are re-tested for aflatoxin by an importing country, some lots accepted in the United States will be rejected by the importing country's aflatoxin testing plan. Computer models were developed to determine the effects of decreasing the final accept level of the U.S. testing plan on the number of lots accepted and rejected in the United States and the number of exported lots accepted and rejected by The Netherlands testing plan. Decreasing the final accept level of the U.S. testing plan from 25 to 5 ppb increased the number of lots rejected in the United States by 371% while reducing the number of exported lots rejected by 51%. For every additional 8.3 lots rejected in the United States, one less export lot will be rejected.     

Comparison of two ELISA screening tests with liquid chromatography for determination of aflatoxins in raw peanuts

A study was conducted to evaluate the performance of 2 enzyme-linked immunosorbent assays (ELISA) for rapidly screening samples of peanuts for the presence of aflatoxin. The EZ-Screen Quick Card Test and the Afla-10 Cup Test were compared with liquid chromatography in duplicate analyses of common extracts of peanuts contaminated in the range of 0-70 ppb (ng/g). Each assay properly identified 95% of samples containing no detectable aflatoxin as negative and greater than 97% of samples containing greater than 10 ppb aflatoxin as positive. The card test, which had a 20 ppb detection threshold, identified as positive 32 of 34 samples in the 11-20 ppb range. This indicates that the card test might actually have a detection threshold closer to 10 ppb. Most of the errors associated with the assays occurred on samples containing less than 10 ppb aflatoxin. The cup and card tests identified 76 and 67% of the samples, respectively, as negative, in the range of 4-10 ppb. For samples either negative or contaminated above their detection thresholds for the assays, the methods are well suited for use as rapid screening tests.     

Minicolumn detection method applied to almonds: collaborative study.

The minicolumn screening method for aflatoxins was collaboratively tested on naturally contaminated almonds. The nuts were extracted, and the extract was cleaned up and applied to a Velasco-type minicolumn. This permits the detection of total aflatoxins (B1, B2, G1, G2) as a fluorescent band on the Florisil layer of the column. The results of 20 collaborators are presented. Samples containing 0, 2, 5, 10, and 25 ng aflatoxin/g were analyzed. Ninety-six per cent of the samples containing 5--25 ng total aflatoxins/g and 83% of the negative samples were correctly identified. The method has been adopted as official first action for detection of total aflatoxin levels of greater than or equal to 5 ng/g.     

Reaction to a paper by Whitaker and Dickens on aflatoxin testing plans for shelled peanuts in the U.S. and the export market

The present paper examines a technical paper of Whitaker and Dickens on aflatoxin testing plans that discusses (without a literature reference) a testing plan used in The Netherlands. However, this testing plan has never been in operation. We present the current situation in The Netherlands with respect to legislation and sampling plans on aflatoxin, which has fairly important consequences for the results of the simulation study of Whitaker and Dickens. It is shown that the percentage of rejected U.S.-exported lots in The Netherlands would increase from 16% to 27% based on the actual testing plan in The Netherlands. The need for international harmonization of testing, and the role of Codex Allmentarius is also emphasized.     

Comparison of an ELISA-based screening test with liquid chromatography for the determination of aflatoxins in corn.

An enzyme-linked immunosorbent assay (ELISA) screening test (CITE PROBE) was compared to liquid chromatography (LC) for the determination of aflatoxins in naturally contaminated corn samples. The CITE PROBE, with a positive/negative cutoff of 5 ng/g aflatoxin B1, was correct (based on LC results) on 47 of 51 samples. Two of the incorrect responses by the CITE PROBE were false positives on samples containing 4.4 ng/g and 4.1 ng/g aflatoxins by LC. Another incorrect response was a false negative on a sample containing 5.5 ng/g aflatoxins by LC. The fourth incorrect response was a false positive on a sample containing 1.9 ng/g aflatoxins by LC. On the basis of these results, the CITE PROBE was determined to be a reliable screening method for the detection of greater than or equal to 5 ng/g aflatoxins in corn.     

Negative ion chemical ionization mass spectrometric method for confirmation of identity of aflatoxin B1: collaborative study

An interlaboratory study of a negative ion chemical ionization mass spectrometric (MS) confirmation procedure for aflatoxin B1 was conducted in laboratories in the United States, England, and West Germany. Twelve partially purified, dry film extracts from naturally and artificially contaminated roasted peanuts, cottonseed, and ginger root containing varying quantities of aflatoxin B1 were distributed to the participating laboratories. The extracts required additional cleanup before MS analysis, using either an acidic alumina column and preparative thin layer chromatography (TLC) or a 2-dimensional TLC procedure. Recovery of purified aflatoxin B1 was influenced by the degree of recovery of sample from acid alumina and/or the TLC plate and incomplete elution of aflatoxin B1 from silica gel. Factors affecting MS confirmation included the purity and recovery of aflatoxin and MS instrument sensitivity. Aflatoxin B1 identity was confirmed in 19.5, 90.9, and 100% of samples containing less than 5, 5-10, and greater than 10 ng aflatoxin B1/g product, respectively, by solid probe introduction using full mass scans. The MS method has been adopted official first action.     

Enzyme-linked immunosorbent assay of aflatoxins B1, B2, and G1 in corn, cottonseed, peanuts, peanut butter, and poultry feed: collaborative study

A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for aflatoxins at 20 ng/g was studied by 12 collaborators. Test samples of peanut butter were extracted by blending with methanol-water-hexane (55 + 45 + 100) and heating the test extracts on a steam bath; test samples of the other commodities were extracted by blending with methanol-water (80 + 20). All test extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of less than 30%. Each diluted filtrate was applied to a cup containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin B1-peroxidase conjugate was added, the cup was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. The test sample was judged to contain greater than or equal to 20 ng aflatoxins/g when, after exactly 1 min, no color was observed on the filter; when a blue or gray color developed, the test sample was judged to contain less than 20 ng aflatoxins/g. All collaborators correctly identified naturally contaminated corn and raw peanut positive test samples. No false positives were found for controls containing less than 2 ng aflatoxins/g. The correct responses for positive test samples spiked at levels of 10, 20, and greater than or equal to 30 ng aflatoxins/g (the ratio of B1:B2:G1 was 10:1:3) were 52, 86, and 96%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Gas chromatographic determination of N-nitrosamines, aromatic amines, and melamine in milk and dairy products using an automatic solid-phase extraction system

A reliable analytical method was presented for the simultaneous determination of six N-nitrosamines, nine aromatic amines, and melamine in milk and dairy products using gas chromatography coupled with mass spectrometry. The sample treatment includes the precipitation of proteins with acetonitrile, centrifugation, solvent changeover by evaporation, and continuous solid-phase extraction for cleanup and preconcentration purposes. Samples (5 g) containing 0.15-500 ng of each amine were analyzed, and low detection limits (15-130 ng/kg) were achieved. Recoveries for milk and dairy products samples spiked with 1, 10, and 50 μg/kg ranged from 92% to 101%, with intraday and interday relative standard deviation values below 7.5%. The method was successfully applied to determine amine residues in several milk types (human breast, cow, and goat) and dairy products.     

Fluorometric measurement of aflatoxin adsorbed on florisil in minicolumns.

Filter fluorometers have been adapted to measure the fluorescence intensity of aflatoxin absorbed on a Florisil layer in minicolumns. The relationship between concentration and intensity is near linear in the aflatoxin range from 10 to 100 ng. Although individual aflatoxin fractions cannot be resolved, since the measure is one of total intensity, fluorometric measurements advance the minicolumn screening procedure to a semiquantitative level. The detection of 1 ng aflatoxin B1 is well within the limits of a filter fluorometer with a photomultiplier detector. A precision, expressed as per cent coefficient of variation, ranging from 1.2 to 4.2%, was obtained for standard B1 columns.     

A rapid aflatoxin B1 ELISA: development and validation with reduced matrix effects for peanuts, corn, pistachio, and Soybeans

Among the competitive ELISAs for aflatoxins that have been described, few have been adequately validated for reduced matrix effects. Using an aflatoxin B(1) (AFB(1))-specific polyclonal antibody (produced from AFB(1)-oxime conjugated to bovine serum albumin (BSA)) and AFB(1)- and AFB(2)-enzyme conjugates, four direct competitive ELISAs based on 96-microwell plates (two standard assays and two rapid assays) were developed, paying special attention to producing a robust assay relatively free of interferences for a range of agricultural products. The antibody was AFB(1)-specific, detecting only AFB(1) in a mixture of four aflatoxins (AFB(1), AFB(2), AFG(1), and AFG(2)), but showed significant cross-reaction with AFG(1) (57-61%) when an individual compound was tested. Standard assays (long assays) exhibited higher sensitivities than rapid assays (short assays) with IC(50) values of 12 +/- 1.5 and 9 +/- 1.5 microg/kg in sample (with 1 in 5 dilution of sample extract) for AFB(1) and AFB(2)-enzyme conjugates, respectively. These assays have narrower detection ranges (7.1-55.5 microg/kg in sample) and required dilution of sample extracts to overcome solvent and matrix interferences, making these assays less ideal as analytical methods. Rapid assays exhibited IC(50) values of 21.6 +/- 2.7 and 12 microg/kg in sample for AFB(1)- and AFB(2)-enzyme conjugates, respectively. These assays have ideally broader detection ranges (4.2-99.9 microg/kg in sample) and showed no methanol effects up to 80% with significantly reduced matrix interferences as a result of the shorter incubation times and increasing the amounts of enzyme conjugate used. Therefore, the rapid assays were formatted to perform without a need for extract dilution. The rapid assays can be completed within 15 min, potentially suitable for receival bays where quick decision-making to segregate low and high contamination is critical. Further validation using the rapid assay with AFB(1)-enzyme conjugate indicated relatively good recoveries of AFB(1) spiked in corn, peanuts, pistachio, and soybeans, which were free from significant matrix effects. It can be concluded that this rapid assay would be suitable for monitoring aflatoxin AFB(1) at current legal maximum residue limits of 10 microg/kg in food such as corn, peanuts, pistachio, and soybeans.