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Four laboratories participated in a mini-collaborative study of AOAC Official Method 971.22, Standards for Aflatoxins, Thin-Layer Chromatographic Method, to extend the method to 3 replacement solvents for benzene for calibration of standard aflatoxin solutions. Triplicate test sample vials, each containing 25 micrograms of the respective aflatoxin for each of the 4 aflatoxins and for each of the solvents, were prepared and sent to each collaborator. The collaborators dissolved the aflatoxin in each vial in 2 mL solvent, measured the UV spectrum, and reported the absorptivity maxima near 350 nm. The concentrations of the aflatoxins in the test samples were determined by dissolving identical test samples in benzene-acetonitrile (98 + 2) and following the procedure described in AOAC Official Method 971.22. These concentrations were, in turn, used to determine the molar absorptivities in the other 3 solvents (see Table 1). AOAC Official Method 971.22 has been modified to extend its applicability to 3 replacement solvents for benzene for calibration of standard aflatoxin solutions.

Broiler breeder hens were fed diets amended with 0 and 10 mg/kg (Trial 1) or 0, 0.2, 1, or 5 mg/kg (Trial 2) of aflatoxin (AF). Fertile eggs collected during 14 d of AF feeding were examined for AF residues. Various immunological endpoints were examined in chicks hatched from these eggs. Eggs collected at 7 d of AF feeding (Trial 1) had 0.15 to 0.48 ng/g of AFB1 and 0.22 to 0.51 ng/g of aflatoxicol, whereas eggs collected at 14 d of AF feeding had 0.05 to 0.60 ng of AFB1/g and 0.19 to 1.20 ng of aflatoxicol/g. In both trials, AF dietary exposure resulted in embryonic mortality and reduction in hatchability compared to controls. The AF progeny chicks in Trial 2 had total anti-SRBC antibodies similar to the controls during the primary antibody response. However, at 5 and 7 d after secondary SRBC injection, the antibody levels in the 1 and 5 mg/kg AF groups were lower than those of controls. Depression in anti-Brucella abortus antibodies occurred only in chicks from the 5 mg/kg AF group. Furthermore, phagocytosis of SRBC and reactive oxygen intermediate production by macrophages from AF progeny chicks were reduced as compared with the control chicks. The findings of this study imply that the progeny chicks from hens consuming a AF-amended diet may be increasingly susceptible to disease owing to suppression of humoral and cellular immunity.

A total of 1019 analytical precision estimates obtained from method-performance (collaborative) studies for mycotoxins published through 1991 were sorted by type of variance measurement, type of analytical method, and type of agricultural commodity. Precision estimates for total aflatoxin were sorted into 2 precision measurements (among-laboratories and within-laboratory), 3 analytical methods (thin-layer chromatography [TLC], liquid chromatography [LC], and enzyme-linked immunosorbent assay [ELISA]), and 11 agricultural commodities. Sufficient data existed to study the analytical variability (precision) associated with 36 sorted combinations (of a possible 66). In all but one combination (within-laboratory, barley, and TLC), the variance (V) was a function of total aflatoxin concentration (C). A power function of the form V = aCb, where a and b are constants, describes the relationship between variance and aflatoxin concentration. The coefficients a and b were determined from regression analysis. When results were pooled across all agricultural commodities, LC had the lowest analytical variability while ELISA had the highest. For a given method, among-laboratories variability was approximately double the within-laboratory variability. These analytical variability estimates can be coupled with previously determined variability estimates of sampling and sample preparation to determine the performance associated with specific test procedures used to inspect agricultural commodities for aflatoxin.

An AOAC/IUPAC collaborative study was conducted to evaluate the effectiveness of a multifunctional column for the determination of aflatoxins. The test portion is extracted with acetonitrile-water (9 + 1), the extract is filtered, and the filtrate is passed through the column. The aflatoxins in the eluate are determined by reversed-phase liquid chromatography after derivatization with trifluoroacetic acid. Naturally contaminated corn, almonds, Brazil nuts, peanuts, and pistachio nuts spiked with total aflatoxins at 5, 10, 20, and 30 ng/g were sent to 12 collaborators in the United States, Denmark, France, Japan, and Switzerland. Eleven collaborators completed the study. Average recoveries of total aflatoxins for each spike level for the various commodities (excluding Brazil nuts at 5 ng/g) were 93, 97, 95, and 95%, respectively; the repeatability relative standard deviation (RSDr) ranged from 6.0 to 23.2% and the reproducibility relative standard deviation (RSDR) ranged from 12.0 to 69.4%. The multifunctional column coupled with a liquid chromatographic method for determination of aflatoxins in corn, almonds, Brazil nuts, peanuts, and pistachio nuts has been adopted first action by AOAC INTERNATIONAL.

An interlaboratory study of a solvent-efficient thin-layer chromatographic (TLC) method for the determination of aflatoxins B1, B2, G1, and G2 was conducted in laboratories located in the United States, France, Tunisia, and Denmark. Eighteen artificially contaminated samples plus blanks of raw peanuts and peanut butter and corn containing varying amounts of aflatoxins B1, B2, G1, and G2 were distributed to participating laboratories. The method consists of elements of the U.S. Food and Drug Administration (FDA), Contaminants Branch (CB) (AOAC Method 968.22) and FDA, Best Foods (BF) (AOAC Method 970.45) methods with reduced requirements for solvents. Participating laboratories used either visual or densitometric techniques during the final determinative step. Statistical analysis of the data was performed to determine or confirm outliers and to compute repeatability and reproducibility of the method using either visual or densitometric techniques for the determinative step. Reported results from laboratories using a densitometer showed that, for corn, the relative standard deviation for repeatability (RSDr) for aflatoxin B1 ranged from 56.6 to 41.7% for contamination levels ranging from 5 to 50 ng/g. For raw peanuts and peanut butter, the RSDr values for aflatoxin B1 ranged from 21.3 to 37.3% and 65.9 to 42.1%, respectively, for the contamination levels ranging from 5 to 25 ng/g. RSDr ranges for aflatoxins B2, G1, and G2 were similar. For reproducibility (R), the RSDR ranges for aflatoxin B1 were 41.7-56.6%, 56.6-84.8%, and 26.4-37.3% for corn, peanut butter, and raw peanuts, respectively. Average recoveries for all aflatoxins at all levels were 95.3, 139.0, and 95.6% for corn, peanut butter, and raw peanuts, respectively. When analysts determined aflatoxin concentrations in corn by visual comparison to standards, the RSDr values for aflatoxin B1 were 47.8-11.4% for contamination levels ranging from 5 to 50 ng/g. For raw peanuts and peanut butter, the RSDr values for aflatoxin B1 were 76.3-12.6% and 33.4-8.8%, respectively, for the contamination levels ranging from 5 to 25 ng/g. RSDr values for aflatoxins B2, G1, and G2 were similar. The RSDR values for aflatoxin B1 were 34.6-90.2%, 45.5-59.3%, and 31.8-78.3% for corn, peanut butter, and raw peanuts, respectively. Average recoveries for all aflatoxins at all levels were 111.0, 157.6, and 92.3% for corn, peanut butter, and raw peanuts, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

An AOAC/IUPAC (International Union of Pure and Applied Chemistry) collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column for the determination of aflatoxin. The test portion is extracted with methanol-water (7 + 3), filtered, diluted to less than 30% methanol with water, and applied to the affinity column. The column is washed with water and the concentrated aflatoxins are eluted with methanol. Total aflatoxins are determined by solution fluorometry with bromine (SFB), and individual toxins are determined by reverse-phase liquid chromatography with postcolumn derivatization with iodine (PCD). Corn naturally contaminated with aflatoxins, and peanuts, peanut butter, and corn containing added aflatoxins (B1:B2:G1:G2 = 7:1:3:1) were sent to 24 collaborators in the United States, France, Canada, and the Republic of South Africa. Twelve collaborators used the SFB method, 9 used the PCD method, and 3 used both SFB and PCD methods. Twenty collaborators completed the study (10 used the SFB method, 7 used the PCD method, and 3 used both SFB and PCD methods). Test portions were spiked at 10, 20, and 30 ng/g. For SFB analyses, recoveries of total aflatoxins were 123, 105, and 107%, respectively; the relative standard deviation for repeatability (RSDr) ranged from 11.75 to 16.57%, and the relative standard deviation for reproducibility (RSDR) ranged from 10.97 to 33.09%. For PCD analyses, recoveries were 81, 81, and 83%, respectively; the RSDr ranged from 5.20 to 17.22%, and the RSDR ranged from 4.68 to 50.77%. The RSDr for aflatoxins B1 and G1 for spiked test portions ranged from 5.45 to 23.55%, and the RSDR ranged from 4.21 to 57.28%.(ABSTRACT TRUNCATED AT 250 WORDS)

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 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)

Six samples each of wheat, corn, lentils, beans, fenugreek, peanuts, and cottonseed cake from various areas of Egypt were analyzed for aflatoxins both at the time of collection and after 12 months' storage. Aflatoxin was found at low levels (3 to 12 ppb total aflatoxins in 14 of 42 samples, as follows: 1 sample each of corn, lentils, and beans; 2 peanut samples; 3 fenugreek samples; and 6 cottonseed cake samples. None of the samples contained aflatoxins above 11.7 ppb.

The thin layer chromatographic method of Trucksess et al. for aflatoxin B1 in eggs was collaboratively studied. Each collaborator analyzed 3 known practice samples and 9 unknown samples containing added aflatoxin B1 at 0, 0.05, 0.10, and 0.30 ng/g. For 9 collaborators, recoveries for the 3 positive levels were: 0--0.13 ng/g (average 98%, coefficient of variation (C.V.) 83%), 0.05--0.18 ng/g (average 102%, C.V. 36%, and 0.11--0.42 ng/g (average 93%, C.V. 31%), respectively. The method has been adopted as official first action.

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.

The Holaday-Velasco method and a modified Holaday method have been compared. The former method combines the speed and simplicity of the Holaday extraction and cleanup with the sensitivity of the minicolumn originally described by Velasco. The combination method has been approved by the AOAC and the AACC for determining aflatoxin in corn. The Holaday method was modified by substituting toluene for benzene in the solvent partition, and methylene chloride for chloroform in the minicolumn development to eliminate use of hazardous solvents. The neutral alumina in the Holaday minicolumn was changed from activity V to activity III to provide a more stable column. At aflatoxin levels in raw peanuts of 13-20 ng/g, the presence of aflatoxin was missed by the modified Holaday method in 4 analyses (3 laboratories) of 42 reported. There were no misses in this contamination range by the Holaday-Velasco method. There were no misses by either method with samples containing greater than 20 ng total aflatoxins/g. Analysis of uncontaminated raw peanuts by the modified Holaday method resulted in 2 false positives of 14 reports; the Holaday-Velasco method produced no false positive reports from 15 analyses of uncontaminated peanuts. The Holaday-Velasco method was adopted official first action for peanuts.

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.

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)