Limited survey of deoxynivalenol in wheat and corn in the United States

A limited survey was conducted over a 2-year period to determine the incidence and levels of deoxynivalenol (DON) in corn and wheat grown in selected areas of the United States. Samples of corn (198) and wheat (247) were collected and analyzed by a gas chromatographic method. Sixty-six percent of the corn samples collected in 1984 and 30% of the corn samples collected in 1985 contained DON. The average concentration of DON in corn, by state, ranged from 0.11 to 1.20 micrograms/g; the maximum concentration was 2.47 micrograms/g. Only 2 of the 247 samples of wheat contained DON at a concentration greater than 2 micrograms/g, which is the level of concern suggested by the Food and Drug Administration for wheat entering the milling process for human consumption.     

Simultaneous analysis of nivalenol and deoxynivalenol in cereals by liquid chromatography

A sensitive method is described for determination of nivalenol (NIV) and deoxynivalenol (DON) in cereals by using reverse phase liquid chromatography and UV detection at 222 nm. The sample is extracted with acetonitrile-water (85 + 15) and an aliquot is purified by passage through a combined column of cation exchange resin and alumina-carbon (20 + 1). Analysis at this stage is possible with some samples but the method recommends passing an aliquot through a carbon minicolumn after evaporation and solubilization in methanol. Interference from coextracted compounds at this point is negligible. Recoveries of both NIV and DON from spiked extracts taken through the full method were in the range 83-94%. The relative standard deviation, based on 5 replicate determinations from each of 2 corn samples, was approximately 5% for both NIV and DON. With a 10 microL injection, the minimum contamination (3 X signal/noise ratio) able to be detected in cereal samples was about 0.015 micrograms NIV/g and 0.05 micrograms DON/g. The cleaned up extracts are also suitable for analysis by gas chromatography.     

Enzyme-linked immunosorbent assay for deoxynivalenol in corn and wheat

The availability of antibody against deoxynivalenol (DON) triacetate (Tri-Ac-DON) has enabled development of a direct enzyme-linked immunosorbent assay (ELISA) and an indirect ELISA for DON in corn and wheat. In both assays, DON is extracted from the sample with acetonitrile-water, reacted with acetic anhydride to form Tri-Ac-DON, and diluted in phosphate buffer for analysis. Direct ELISA was found to be the more sensitive procedure. Fewer interferences are evidenced, and the assay is less time consuming than is indirect ELISA. For direct ELISA, recovery of 10-1000 ppb DON added to corn and wheat was 100% (SD 15, CV 15%) and 102.1% (SD 12.2, CV 11.9%), respectively. For indirect ELISA, overall recovery of 10-1000 ppb DON added to wheat was 121.5% (SD 39.5, CV 32.5%); in the higher concentration range (500-1000 ppb), recovery was 105% (SD 18, CV 17%). The minimal detection level for DON was around 10 ppb. Analysis of 7 naturally contaminated samples for DON showed that the ELISA results agreed well with those obtained by radioimmunoassay and thin-layer chromatography.     

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.     

Thin layer chromatographic method for determination of deoxynivalenol in wheat: collaborative study

A collaborative study of a rapid method for the determination of deoxynivalenol (DON) in winter wheat was successfully completed. The method involves sample extraction with acetonitrile-water (84 + 16), cleanup using a disposable column of charcoal, Celite, and alumina, and detection by thin layer chromatography after spraying with an aluminum chloride solution. Each of the 15 collaborators analyzed 12 samples, 2 of which were naturally contaminated, and 10 to which DON was added, in duplicate, at levels of 0, 50, 100, 300, and 1000 ng/g. Average recoveries of DON ranged from 78 to 96% with repeatabilities of 30-64% and reproducibilities of 33-87%. The results of the study show that false positives were not a problem and that all of the analysts could detect DON at the 300 ng/g level or higher. The method has been adopted official first action.     

Liquid chromatographic determination of alpha-zearalenol and zearalenone in corn: collaborative study

The liquid chromatographic determination of alpha-zearalenol and zearalenone in corn was collaboratively studied. Each of 13 collaborators received 7 corn samples; 2 were blanks and 5 were spiked to contain 50, 100, and 200 ng alpha-zearalenol/g and 50, 100, 500, 1000, and 4000 ng zearalenone/g. Four sets (including blanks) of blind duplicates were included in the study. Five naturally contaminated corn samples (one in duplicate) were also provided. All collaborators detected both mycotoxins at 50 ng/g. Average recoveries reported by all collaborators ranged from 81.9% at 200 ng/g to 100.3% at 50 ng/g for alpha-zearalenol and from 77.8% at 1000 ng/g to 123% at 50 ng/g for zearalenone. Three collaborators reported false positives for both alpha-zearalenol and zearalenone. The within-laboratory CV values based on blind duplicates were 22.6% for alpha-zearalenol and 31.4% for zearalenone. The CV values based on laboratory-sample interaction were 25.6 and 33.8% for alpha-zearalenol and zearalenone, respectively. The CV values for naturally contaminated samples (including duplicates) were 47.0% for alpha-zearalenol and 37.7% for zearalenone. The method has been adopted official first action.     

Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels

Maize (Zea mays) and wheat (Triticum aestivum) collected in the foothills of the Nepal Himalaya Mountains were analyzed for Fusarium species and mycotoxins: fumonisins, nivalenol (NIV), and deoxynivalenol (DON). Predominant species were Gibberella fujikuroi mating population A (F. moniliforme) in maize and F. graminearum in maize and wheat; G. fujikuroi mating population D (F. proliferatum), F. acuminatum, F. avenaceum, F. chlamydosporum, F. equiseti, F. oxysporum, F. semitectum, and F. torulosum were also present. Strains of G. fujikuroi mating population A produced fumonisins, and strains of F. graminearum produced NIV or DON. By immunoassay or high-performance liquid chromatography, fumonisins were >1000 ng/g in 22% of 74 maize samples. By immunoassay or fluorometry, NIV and DON were >1000 ng/g in 16% of maize samples but were not detected in wheat. Fumonisins and DON were not eliminated by traditional fermentation for producing maize beer, but Nepalese rural and urban women were able to detoxify contaminated maize by hand-sorting visibly diseased kernels.     

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.     

Deoxynivalenol Content of Cereal Grain from Naturally Infected and Artificially Inoculated Plants in Field Trials in Norway

The Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3-acDON) were determined in grain samples from naturally infected and Fusarium culmorum inoculated plants in field experiments in Norway during 1992–1996. The mean DON content in trials with inoculated plants was 11.8 μg/g in spring oats, 11.3μg/g in winter wheat, 28.9 μg/g in spring wheat and 31.4 μg/g in spring barley. In the natural infection trials the mean DON content was 0.32 μg/g in spring oats, 0.22μg/g in winter wheat, 1.48μg/g in spring wheat and 0.54 μg/g in spring barley. Only small differences in DON content were observed among cultivars, and significant differences were found only in winter wheat in the inoculation trials, and in spring wheat in the natural infection trials. A significant correlation was observed between the 3-acDON and DON contents in the inoculated trials in all grain species, the mean ratio of 3-acDON to DON ranging from 0.011 in wheat to 0.071 in oats.     

Liquid chromatographic method for determination of citreoviridin in corn and rice

Citreoviridin, a neurotoxic mycotoxin, has been found as a natural contaminant in corn left unharvested in the southeastern United States and in rice of several Asian countries, including Japan. A reliable analytical method for the quantitative determination of citreoviridin in corn and rice is described. Corn or rice is extracted with dichloromethane, and the extract is partially purified on silica and amino solid-phase extraction (SPE) columns. The extract is analyzed for citreoviridin by normal-phase liquid chromatography, using a mobile phase of ethyl acetate-hexane (75 + 25) at 1.5 mL/min and a fluorescence detector to measure the yellow fluorescence (388 nm excitation, 480 nm emission). With a 100 microL injection loop, the relationship between concentration and injection volume is linear for 20-60 microL injections. Recoveries of citreoviridin added to yellow corn at 10-50 ng/g were 91.0-96.9%; recoveries from white corn (10-50 ng/g added) were 96.8-102.8%. Recoveries of 5000 ng/g added to white corn were 89.0%, indicating that heavily contaminated samples can be assayed by the method. Minimum detection limits were 10 ng for citreoviridin standard and 2 ng/g for citreoviridin added to corn. White rice fermented with Penicillium citreo-viride (1524 ppm) was mixed with and serially diluted with uncontaminated ground corn to obtain citreoviridin-contaminated corn (ca 25 ppb). When the samples were assayed by the method, a mean level of 24.4 +/- 1.65 ppb (6.5% coefficient of variation) was obtained. Four fermented rice food samples and 3 commercial rice samples were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of two immunochemical methods with thin-layer chromatographic methods for determination of aflatoxins

Three different methods were compared for the determination of total flatoxins in corn and peanuts naturally contaminated with aflatoxins and in corn, peanuts, cottonseed, peanut butter, and poultry feed spiked with aflatoxins B1, B2, and G1. The 3 methods were an enzyme-linked immunosorbent assay (ELISA) screening test; a monoclonal antibody-affinity column-solid-phase separation method; and the AOAC official thin-layer chromatography (TLC) methods for all except poultry feed, for which Shannon's TLC method for mixed feed was used. The ELISA test is designed to provide only positive results for total aflatoxins at greater than or equal to 20 ng/g or negative results at less than 20 ng/g. The affinity column separation is coupled with either bromination solution fluorometry to estimate total aflatoxins or liquid chromatography (LC) to quantitate individual aflatoxins. Fluorodensitometry was used to determine aflatoxins in commodities analyzed by the TLC methods. The LC and TLC results were in good agreement for all the analyses. The results for the affinity column using bromination solution fluorometry were similar except those for cottonseed, which were about 60% higher. The ELISA screening method correctly identified naturally contaminated corn and peanut positive samples. No false positives were found for controls. The correct response for spiked corn, raw peanuts, peanut butter, and cottonseed at greater than or equal to 20 ng aflatoxins/g was about 90%. The correct response for spiked poultry feed at greater than or equal to 20 ng aflatoxins/g was about 50%.     

Co-occurrence of ochratoxin A and citrinin in cereals from Bulgarian villages with a history of Balkan endemic nephropathy

Cereal samples were collected in 1998 from Bulgarian villages without [control village (C), n = 20] or with [endemic villages (E); E1, n = 21; E2, n = 30; E3, n = 23] a history of Balkan endemic nephropathy (BEN). Sampling included foods (wheat, corn) and feeds (barley, oats, wheat bran). Analysis of ochratoxin A and citrinin was done by enzyme immunoassays (EIA), with detection limits of 0.5 and 5 ng/g, respectively. Ochratoxin A-positive results were confirmed by HPLC after immunoaffinity chromatography. Highest toxin levels were found in wheat, wheat bran, and oats. For ochratoxin A, the percentages of positives were 35% (C), 29% (E1), 30% (E2), and 47% (E3), the mean/median values of positives were 1.5/1.3 ng/g (C), 11/1.6 ng/g (E1), 18/1.6 ng/g (E2), and 3.5/1.5 ng/g (E3). For citrinin, 5.0% (C), 14% (E1), 3.3% (E2), and 13% (E3) were positive, and the mean/median values were 6.1/6.1 ng/g (C), 180/83 ng/g (E1), 10/10 ng/g (E2), and 84/20 ng/g (E3). Highest concentrations of ochratoxin (maximum = 140 ng/g) and citrinin (maximum = 420 ng/g) were found in samples from endemic villages. Co-contamination with ochratoxin A and citrinin was found for one sample (14% of positives) from village C and for six samples (22% of positives) from villages E1-E3. Citrinin levels in these samples were 2-200 times higher than those of ochratoxin A.     

Screening and quantitation of ochratoxin A in corn, peanuts, beans, rice, and cassava

To answer the need for simple, economical, rapid methods for mycotoxins, a procedure for screening and quantitation of ochratoxin A was developed. A methanol-aqueous KCl extraction is used, followed by cleanup with clarifying agents and partition into chloroform. Part of the chloroform extract is used for screening and the other part for quantitation by thin layer chromatography (TLC). The screening procedure takes 40 min, using a silica gel/aluminum oxide minicolumn developed for this purpose. The limits of detection are 80 and 10 micrograms/kg, respectively, for minicolumn screening and TLC quantitation. Ammonium sulfate is efficient in cleaning samples of corn and cassava; cupric sulfate is better with peanuts, beans, and rice. Tests were conducted on triplicate spiked samples of yellow corn meal, raw peanuts, dried black beans, polished rice, and cassava flour at different levels (400, 200, 80, 40, and 10 micrograms/kg). Recoveries ranged from 86 to 160% and the coefficients of variation ranged from 0 to 26%.     

Radioimmunoassay of deoxynivalenol in wheat and corn

With the availability of antibody against deoxynivalenol triacetate (DON-triacetate), a radioimmunoassay (RIA) for DON in wheat was developed. DON is extracted from the sample with acetonitrile-water (84 + 16), defatted with hexane, and then reacted with acetic anhydride to form DON-triacetate. The reaction mixture is loaded onto a C-18 cartridge to remove excess reagents and impurities. Acetylated DON is eluted from the cartridge with 50% methanol in water, and then analyzed by radioimmunoassay utilizing antiserum against DON-triacetate and tritiated DON-triacetate. Overall recovery for DON added to wheat between 50 and 5000 ppb was 86% with a standard deviation of 7% and coefficient of variation of 8%. The limit of detection for DON was about 20 ppb. Analysis of 12 naturally contaminated wheat, corn, and mixed feed samples for DON revealed that RIA results agreed well with thin layer chromatographic analyses performed by other laboratories.     

Simple, rapid, and inexpensive cleanup method for quantitation of aflatoxins in important agricultural products by HPLC

A chemical cleanup procedure for low-level quantitative determination of aflatoxins in major economically important agricultural commodities using HPLC has been developed. Aflatoxins were extracted from a ground sample with MeOH/H2O (80:20, v/v), and after a cleanup step on a minicolumn packed with Florisil, aflatoxins were quantified by HPLC equipped with a C18 column, a photochemical reactor, and a fluorescence detector. Water/MeOH (63:37, v/v) served as the mobile phase. Recoveries of aflatoxins B1, B2, G1, and G2 from peanuts spiked at 5, 1.7, 5, and 1.7 ng/g were 89.5+/-2.2, 94.7+/-2.5, 90.4+/-1.0, and 98.2+/-1.1, respectively (mean+/-SD, %, n=3). Similar recoveries, precision, and accuracy were achieved for corn, brown and white rice, cottonseed, almonds, Brazil nuts, pistachios, walnuts, and hazelnuts. The quantitation limits for aflatoxins in peanuts were 50 pg/g for aflatoxin B1 and 17 pg/g for aflatoxin B2. The minimal cost of the minicolumn allows for substantial savings compared with available commercial aflatoxin cleanup devices.     

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)     

Simple and sensitive determination of o-phenylphenol in citrus fruits using gas chromatography with atomic emission or mass spectrometric detection

In this work, a simple and sensitive method for the analysis of the pesticide o-phenylphenol (OPP) on citrus fruits was developed. OPP is extracted with dichloromethane by ultrasonication and derivatized with ferrocenecarboxylic acid chloride. Using ferrocene as a label, residues of OPP are determined by gas chromatography with atomic emission detection in the iron selective mode or with mass spectrometric detection. Sample cleanup is simple and rapid and merely involves a removal of excess reagent on an alumina minicolumn. The method detection limit is 2 ng of OPP/g of fruit, and recoveries from lemon samples fortified at levels of 35 and 140 ng/g are 101 and 106%, respectively. The citrus fruits analyzed (oranges, grapefruits, lemons) contained between 60 ng/g and 0.37 microg/g OPP (RSD = 8-13%), and the results were in good agreement with results obtained when OPP was analyzed using an established HPLC-FLD method. Several alcohols could also be identified in the fruit peel.     

Liquid chromatographic determination and stability of the Fusarium mycotoxin moniliformin in cereal grains

Moniliformin is a mycotoxin produced by Fusarium subglutinans and other Fusarium species. A rapid, liquid chromatographic method for its determination in corn and wheat is described. Samples are extracted in acetonitrile-water (95 + 5); following defatting with n-hexane, an aliquot of the extract is evaporated and cleaned up on small C18 and neutral alumina columns successively. Reverse-phase liquid chromatography (LC) is conducted on a C18 column with 10 or 15% methanol or acetonitrile in aqueous ion-pair reagent as mobile phase, with detection by ultraviolet absorption at 229 and 254 nm. Average recoveries of moniliformin (potassium salt) added to ground corn and wheat at levels of 0.05-1.0 micrograms/g were 80% (n = 20) and 85% (n = 12), respectively, and the limit of detection was ca 0.01-0.18 micrograms/g, depending on LC conditions. Analysis of 24 samples of wheat, 4 samples of rye, and 12 samples of corn showed moniliformin in only 2 corn samples (0.06 and 0.2 micrograms/g). Moniliformin was also detected in a sample of artificially damaged (slashed) corn (0.2 micrograms/g) and selected kernels of corn that were field-inoculated with F. subglutinans and F. moniliforme (50 micrograms/g and 0.5 micrograms/g, respectively). In stability studies, moniliformin (potassium salt, 1 microgram/g) in ground corn and ground wheat heated at 50, 100, and 150 degrees C for 0.5-2 h decomposed moderately, e.g., 55% remained in corn after 0.5 h at 100 degrees C.     

Preparation of deoxynivalenol (vomitoxin) from field-inoculated corn

A process was developed for production of gram quantities of deoxynivalenol (DON) from corn that had been inoculated in the field with Fusarium graminearum and was estimated to contain 400-500 mg DON/kg. Steps in the purification procedure included extraction with methanol-water (1 + 1), partition from an aqueous solution into ethyl acetate by using a hydrophilic matrix, defatting, Florisil column chromatography, methylene chloride-water partition, semipreparative liquid chromatography (LC), and crystallization from ethyl acetate. The average yield of crystalline DON produced by this method (or with minor variations) was 281 mg/kg.     

Thin layer chromatographic determination of deoxynivalenol in processed grain products

The thin layer chromatographic (TLC) method of Trucksess et al. (J. Assoc. Off. Anal. Chem. (1984) 67, 40-43) was modified for the determination of deoxynivalenol (DON) in high-sugar breakfast cereals, corn syrup, and beer. Celite was added to the substrate before extraction with acetonitrile-water (84 + 16). After filtration through an alumina-charcoal-Celite (0.5 + 0.7 + 0.3) column, the filtrate was evaporated to dryness and redissolved in water, which was passed through an octylsilyl reverse phase column. DON was eluted with anhydrous ethyl ether. The residue remaining after the eluate was evaporated to dryness was dissolved in CHCl3-acetonitrile (4 + 1) and chromatographed on AlCl3-impregnated silica gel TLC plates. The blue fluorescent DON spot was quantitated fluorodensitometrically after the TLC plate was heated at 120 degrees C for 7 min. Recoveries of DON added to breakfast cereals at 100, 200, and 400 ng/g levels and to syrup and beer at 50, 100, and 200 ng/g levels averaged 86%. The limit of determination in these products was about 50 ng/g.