High pressure liquid chromatographic determination of aflatoxins in corn.

A high pressure liquid chromatographic (HPLC) method is proposed for determining aflatoxins in corn. The sample is extracted with methanol-10% NaCl (4 + 1), pigments are precipitated with zinc acetate, and the extract is cleaned up on a small (2 g) silica gel column. Aflatoxins in the purified extract are resolved by normal phase HPLC on a microparticulate (10 micrometer) silica gel column with water-saturated chloroform-cyclohexane, acetonitrile solvent, and detected by fluorescence on a silica gel-packed flowcell. The method was compared with chloroform-water extraction of the official CB method on 15 samples of contaminated corn. In 5 of the 6 samples containing aflatoxins B1, B2, G1, and G2, methanol-10% NaCl extracted more aflatoxin than did cloroform-water, as measured both by HPLC and by thin layer chromatography. In samples containing only B1 and B2, the 2 extraction solvents were virtually equivalent. Agreement was good between HPLC and TLC for each extraction solvent. Average recovery of aflatoxins B1, B2, G1, and G2 added to yellow cornmeal at 3 levels was greater than 90%.     

Screening method for the detection of aflatoxin, ochratoxin, zearalenone, penicillic acid, and citrinin.

A modification of the official method for ochratoxins and a screening method for zearalenone, aflatoxin, and ochratoxin is described and expanded to include citrinin and penicillic acid. The method uses 0.5N phosphoric acidchloroform (1+10) in the initial extraction; the extract is divided and eluted from 2 columns to provide a quantitative thin layer chromatographic (TLC) method for aflatoxin and ochratoxin in corn and dried beans. Aflatoxin and zearalenone are eluted from one column and ochratoxin, penicillic acid, and citrinin from the other. Ochratoxin A recoveries are low (50%) in peanuts. Zearalenone, penicillic acid, and citrinin were qualitatively recovered from corn and beans; zearalenone and penicillic acid were recovered from peanuts but citrinin was not. Several TLC solvents were used to separate interferences.     

An analytical survey of aflatoxins in tissues from swine grown in regions reporting 1988 aflatoxin-contaminated corn.

A joint project was undertaken by the Food Safety and Inspection Service (FSIS) and the Agriculture Research Service branches of the U.S. Department of Agriculture to determine the presence of aflatoxins in the U.S. meat supply during a drought year. In 1988, high incidences of aflatoxins occurred in corn grown in regions of the Midwest, Southeast, and South. Six states were identified as having serious aflatoxin contamination in their corn crop: Virginia, North and South Carolina, Texas, Iowa, and Illinois. Swine liver and pillars of diaphragm (muscle) tissues were sampled by federal FSIS Inspectors in plants located in these states. A worstcase sampling plan was conducted. Samples were taken in January 1989 from hogs fed corn soon after harvest and in April 1989 from hogs fed corn originally stored and then fed in the spring. A modification of the official AOAC method for the thin-layer chromatography (TLC) determination of aflatoxins in animal tissue was used to permit quantitation by LC with fluorescence detection. The official AOAC TLC confirmation of identity method was used to confirm all positive samples with B1 concentrations greater than 0.04 ppb and M1 concentrations greater than 0.1 ppb. Sixty samples in the January group and 100 samples in the April group were assayed. Concentrations of aflatoxins B1 and M1 in the first group of pig livers ranged from 0.04 to 0.06 ppb. The identity of aflatoxin B1 was confirmed in all positive samples. Aflatoxin M1 could not be confirmed in any of the positive liver samples because the method was insufficiently sensitive for this aflatoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

A quantitative method for determination of aflatoxin B in roasted corn.

Roasting aflatoxin-contaminated corn will reduce toxin levels. A quantitative analysis for aflatoxin in roasted corn has been developed by modifying a cleanup technique for green coffee extracts approved as official first action by the AOAC. A chloroform extract is partially purified on a Florisil column, and thin layer chromatographic (TLC) plates are developed with methylene chloride-chloroform-isoamyl alcohol-formic acid (81+15+3+1). Recoveries average 101% and the sensitivity limit is 5 ppb aflatoxin B1. A 2-dimensional TLC procedure can also be used to separate the aflatoxins from background interferences.     

Comparison of postcolumn derivatization-liquid chromatography with thin-layer chromatography for determination of aflatoxins in naturally contaminated corn

Quantitation of aflatoxins by liquid chromatography with postcolumn iodine derivatization (LC-PCD) and fluorescence detection was compared with quantitation by the AOAC CB method, 968.22. Thirty-seven naturally contaminated corn samples were ground and then divided. One portion was extracted, and the extract was cleaned up and analyzed by thin-layer chromatography according to the CB method. The second portion was extracted and cleaned up in a similar fashion, but quantitation was by the LC-PCD method. For aflatoxin B1 concentrations ranging from 0 to 150 ng/g, results obtained by the 2 methods were fitted to a linear equation with the LC-PCD results as the dependent variable. The correlation coefficient was 0.99, the intercept was near 0, and the slope was near 1. For aflatoxin B2, the correlation coefficient was 0.97, and the intercept was near 0. However, the slope of the equation relating LC-PCD concentration to TLC concentration was only 0.5. We believe that this lack of equivalence between the methods for determination of aflatoxin B2 is due to overestimation by the TLC method because the low levels present are near the TLC detection limit for B2.     

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.     

Fluorimetric determination of aflatoxin M1 in cheese

A simple and sensitive method is proposed for the determination of aflatoxin M1 in cheese. The ground cheese sample is extracted with acetone-water (3 + 1). Acetone is evaporated under vacuum, and the aqueous phase is passed through a C18 disposable cartridge. After the cartridge is washed with acetonitrile-water (1 + 9), the toxin is eluted with acetonitrile. The extract is then cleaned up on a silica cartridge. Final analysis is performed by 2-dimensional thin layer chromatography (TLC) combined with fluorodensitometry or by liquid chromatography on a reverse phase C18 column with fluorescence detection. Recovery is greater than 90%, and the coefficient of variation is 6% or less. The detection limit is in the range of 10 ng/kg. The identity of aflatoxin M1 is confirmed by formation of the M2a or acetyl-M1 derivative and rechromatography.     

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.     

Comparative evaluation of commercially available aflatoxin test methods

Five qualitative methods and 1 quantitative aflatoxin analytical method were compared with the Holaday-Velasco (HV) minicolumn and thin-layer chromatography (TLC) methods for corn in an evaluation involving 4 U.S. Department of Agriculture Federal Grain Inspection Service (USDA-FGIS) laboratories, 1 laboratory at the University of Georgia, and 1 laboratory at the University of Arizona. Samples analyzed included 1 set of artificially contaminated corn containing both aflatoxin B1 and B2 (ratio of B1:B2 of 92:8), 1 set of artificially contaminated corn containing only aflatoxin B1, and 1 set of naturally contaminated corn. Levels of total aflatoxin tested were 0, 10, 15, 20, 25, 30, and 40 ppb. Results of analysis of these samples with each method evaluated are reported. Chi-square analyses indicated that performance of the Afla-20-Cup, Aflatest, EZ-Screen, OXOID, and SAM-A methods was not statistically different from that of the HV minicolumn. Agri-Screen results were not statistically different from those obtained with TLC.     

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.     

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.     

Aflatoxins and fumonisins in corn from the high-incidence area for human hepatocellular carcinoma in Guangxi, China

A comparative study on the natural occurrence of aflatoxins and Fusarium toxins was conducted with corn samples from high- and low-incidence areas for human primary hepatocellular carcinoma (PHC) in Guangxi, China. In samples from the high-risk area, aflatoxin B(1) was the predominant toxin detected in terms of quantity and frequency, with its concentration ranging between 9 and 2496 microg/kg and an 85% incidence of contamination. Among the samples, 13 (76%) exceeded the Chinese regulation of 20 microg/kg for aflatoxin B(1) in corn and corn-based products intended for human consumption. Significant differences in aflatoxin B(1), B(2), and G(1) and total aflatoxin concentrations in corn between the areas were found (P < 0.05). The average daily intake of aflatoxin B(1) from corn in the high-risk area was 184.1 microg, and the probable daily intake is estimated to be 3.68 microg/kg of body weight/day, 3.20 times the TD(50) in rats. Corn samples from both areas were simultaneously contaminated with fumonisins B(1), B(2), and B(3). Aflatoxin B(1) may play an important role in the development of PHC in Guangxi.     

Rapid thin layer chromatographic method for determining aflatoxin B1, ochratoxin A, and zearalenone in corn

A multimycotoxin thin layer chromatographic method is described for the analysis of corn. Aflatoxins are extracted from the samples with acetonitrile-water, and sodium bicarbonate is added to separate the acidic ochratoxin from zearalenone and aflatoxin B1. After chloroform extraction, 1N NaOH is added to separate zearalenone and aflatoxin B1. The separated mycotoxins are spotted on TLC plates, which are then examined under ultraviolet light. The following recoveries (%) were obtained for corn samples: aflatoxin B1 71, ochratoxin A 87, and zearalenone 85. The limits of detection for the respective mycotoxins were 2, 40, and 200 ppb.     

Time course study of substrate utilization by Aspergillus flavus in medium simulating corn (Zea mays) kernels.

Utilization of the three major corn reserve materials, starch, triglycerides (refined corn oil), and zein (storage protein), by Aspergillus flavus was monitored in vitro over a 7-day fermentation. Medium composition in which proportions of reserve materials initially approximated proportions in mature corn kernels changed little over the first 18 h. Subsequently, hydrolysis of both starch and triglycerides occurred simultaneously, with peak concentrations of glucose and free fatty acids on day 2 of the fermentation period. Fatty acid concentrations dropped relatively rapidly after day 2 but increased again after day 6. Aflatoxin B(1) production increased after 36 h, with a peak at day 4. Aflatoxin B(1) production paralleled fungal biomass production during the exponential growth phase. A. flavus did not appear to preferentially utilize any of the released fatty acids. A number of fungus-specific metabolites were detected, including arabitol, erythritol, mannitol, trehalose, and kojic acid. Mannitol exceeded the other metabolites in concentration, and the timing of mannitol production closely paralleled that of aflatoxin B(1). Kojic acid concentrations peaked at day 6. In contrast to previously described selective use of simple carbohydrates by A. flavus, less discrimination was displayed when faced with utilization of complex substrates such as starch or triglycerides.     

Rapid immunochemical screening method for aflatoxin B1 in human and animal urine

A method has been developed to determine the presence of aflatoxin B1 in the urine of animals (including humans) by utilizing commercial immunochemical kits that can be used in the field. Urine is treated with diatomaceous earth and filtered to clarify the sample; 2-3 ppb aflatoxin B1, corresponding to about 300 ppb in the ingested feed/food, can be detected in the filtered urine without further purification. To improve sensitivity, the urine filtrate is passed through a C18 solid phase column to extract the aflatoxin. The column is washed with acetonitrile-water (15 + 85) and water, aflatoxin B1 is eluted with methanol-water (7 + 3), and water is added to the eluate, which is then tested for aflatoxin with the test kit. The limit of detection is 0.2 ppb, reflecting consumption of 40 ppb or more aflatoxin in the feed/food. When the initial sample volume is adequate, purification through the C18 column step is usually sufficient. For limited sample volumes, the eluate from the C18 column is mixed with water, added to an immunosorbent affinity column, and washed with water to remove excess sample matrix and impurities. Aflatoxin B1 is eluted with acetonitrile. The extract is evaporated under nitrogen and the residue is redissolved in methanol-water (25 + 75). At this purification stage, the limit of detection is reduced to 0.05 ppb.     

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.     

Determination of zearalenone in cornflakes and other corn-based foods by thin layer chromatography, high pressure liquid chromatography, and gas-liquid chromatography/high resolution mass spectrometry

A simple cleanup procedure based on pH adjustments was used to obtain extracts of corn foods. The method gave good recoveries of zearalenone determined by thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). As little as 5 ng zearalenone was detected by TLC, using Fast Violet B Salt as the spray reagent; the lower limit of detection in cornflakes was about 20 microgram/kg. With HPLC on Spherisorb silica (5 micrometer) and detection by fluorescence at an excitation maximum of 310 nm as little as 5 microgram zearalenone/kg cornflakes could be determined. While the TLC method was also applicable to corn chips, cornmeal, popcorn, and frozen corn, an interference was observed in HPLC of the latter 3 products. This interference was separated from zearalenone by adding a second HPLC analytical column (Spherisorb ODS). Gas-liquid chromatography coupled with mass spectrometric single ion monitoring at high resolution, although of limited availability, was shown to be the most sensitive and selective method for determining zearalenone in corn foods. The natural occurrence of zearalenone in a sample of cornflakes (13-20 microgram/kg) was demonstrated by all 3 detection procedures.     

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.     

Thin layer chromatographic confirmation of aflatoxin M1 extracted from milk

Aflatoxin M1 can be confirmed directly on a thin layer plate by reacting the toxin with a mixture of reagents containing p-anisaldehyde. This confirmatory procedure requires only 2 elutions in the same direction using 2 different solvents. The mixture containing p-anisaldehyde is overspotted on M1 after the plate has been developed in toluene-ethyl acetate-ethyl ether-formic acid (25 + 35 + 40 + 5). The plate is heated at 110 degrees C for 10 min and then developed in hexane-acetone-chloroform (15 + 50 + 35). The Rf value of the green fluorescent derivative is less than that of the M1 standard. This confirmatory procedure requires only one-dimensional TLC, so several sample extracts and the standard can be run simultaneously. The minimum detectable quantity of aflatoxin M1 on the TLC plate with this test is 0.3 ng. p-Anisaldehyde reagent solution may also be used as a spray reagent for the confirmation of aflatoxin M1. The procedures described were satisfactory for confirming the mycotoxin in spiked samples of powdered and liquid milk.     

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.