Gas chromatographic method for ethylene dibromide in grains and grain-based products: collaborative study

Nine laboratories analyzed samples of whole grain, intermediate, and ready-to-eat products for ethylene dibromide (EDB) residues. Supplied samples of wheat, rice, and flour contained both fortified and incurred EDB; corn bread mix, baby cereal, and bread contained only fortified EDB. The whole grains and intermediates were analyzed by the same basic procedural steps as in the official method for multifumigants: They were extracted by soaking in acetone-water (5 + 1). The baby cereal and bread were analyzed by a modification of the Rains and Holder hexane co-distillation procedure. EDB was determined by electron capture gas chromatography operated with an SP-1000 column. All products contained 3 different levels of EDB and were analyzed as blind duplicates. Overall mean recoveries ranged from 85.2% for 69.6 ppb to 105.0% for 4.35 ppb, both in baby cereal. Interlaboratory relative standard deviations ranged from 5.7% for 869 ppb in wheat to 20.2% for 69.6 ppb in baby cereal, both fortified. Mean levels of incurred EDB in wheat, rice, and flour were 926.7, 982.0, and 49.9 ppb, respectively; corresponding relative standard deviations were 9.9, 7.7, and 13.1%. The method was adopted official first action.     

A gas chromatography-flame ionization detection method for detection of fusaproliferin in corn

A sensitive and accurate detection method is of great importance in monitoring fusaproliferin levels in foods and animal feeds and evaluating its potential hazard to human and animal health. Several methods have been developed to detect fusaproliferin in cereals and cereal-related products, including thin-layer chromatography, high-performance liquid chromatography, enzyme-linked immunosorbent assay, liquid chromatography-mass spectrometry (MS), gas chromatography (GC), and GC-MS. However, these detection methods either suffer from low sensitivity, need expensive instruments, or are susceptible to interfering substances in the sample matrix. The GC-flame ionization detector method developed herein is sensitive, reliable, and easy to use for detecting fusaproliferin in corn and corn-based samples. Its detection limits were 0.04 ng for standard trimethylsilyl-fusaproliferin and about 5 ppb for fusaproliferin in corn samples. The limits of quantitation of this method were 0.15 ng fusaproliferin/injection and 20 ppb of fusaproliferin in corn samples. The recovery rates of fusaproliferin from corn samples spiked with 200, 1000, and 5000 ppb standard fusaproliferin were 109, 85.7, and 98.9% on average. The repeatability of the method was acceptable when evaluated by the Horwitz equation. Of the tested corn samples, three out of five sweet corn and the three yellow corn samples were found to have low levels of fusaproliferin (9.4-45.3 ppb). A moldy corn sample had a fusaproliferin content of 297 ppb.     

Purge and trap method for determination of ethylene dibromide in whole grains, milled grain products, intermediate grain-based foods, and animal feeds

An improved method has been developed for the determination of ethylene dibromide (EDB; 1,2-dibromoethane) in whole grains, milled grain products, intermediate grain-based foods, and animal feeds. Samples are mixed with water and sparged with nitrogen for 1 h with stirring in a water bath at 100 degrees C. The EDB collected on the adsorbent Tenax TA is eluted with hexane and determined by gas chromatography (GC) with electron capture detection (ECD) and confirmed with Hall electrolytic conductivity detection (HECD) using a second GC column. The highest levels of EDB were also confirmed by full scan GC/mass spectrometry (GC/MS). A total of 24 whole grains, milled grain products, intermediate grain-based foods, and animal feeds analyzed by using this method contained EDB levels up to 840 ppb (wheat). Recoveries from fortified samples ranged from 90 to 105%. Values from this method were compared with those obtained from the acetone soak method; for all 24 samples, this purge and trap method gave equivalent or superior recoveries and detected levels of EDB. Chromatograms for this purge and trap method were clean, enabling a quantitation level of 0.5 ppb to be achieved.     

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.     

Immunochromatography of fusarochromanone mycotoxins

The present paper describes an enzyme-linked immunoassay (ELISA) used in combination with thin-layer chromatography (TLC) and liquid chromatography (LC) for determination of fusarochromanone (TDP) mycotoxins in barley, wheat, and a Fusarium culture grown in rice and corn. The mycotoxins were first extracted from the sample with 100% methanol and subjected to TLC or LC without additional cleanup treatment. Individual fractions eluted from TLC or LC were acetylated, then analyzed by ELISA. Determinations of TDP toxins at levels as low as 0.1 and 0.5 ng were achieved by ELISA in combination with LC and TLC, respectively. The detection limit for TDP-1 in barley and wheat was about 20 ppb by ELISA alone as compared with a detection limit of 5 ppb by a combination of ELISA with either TLC or LC. Overall analytical recovery (% of added) of TDP-1 added to barley and wheat at 5, 10, and 20 ppb of TDP-1 was 106.9 +/- 15.3 and 113.2 +/- 11.6 by LC-ELISA and 108.8 +/- 9.1 and 110.4 +/- 4.9 by TLC-ELISA, respectively. Analysis of extracts obtained from Fusarium equiseti R6137 grown in corn and rice by the combination of TLC and ELISA revealed that diacetyl-TDP was also produced by this fungus in addition to TDP-1 and TDP-2. Comparable results were obtained when fungal extracts were subjected to ELISA, LC, and immunochromatography (i.e., combination of ELISA with either TLC or LC).     

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.     

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.     

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.     

Purge and trap method for determination of fumigants in whole grains, milled grain products, and intermediate grain-based foods

A method developed for the determination of 1,2-dibromoethane in whole grains and grain-based products has been modified and expanded to include 8 other fumigants. Samples are stirred with water and purged with nitrogen for 0.5 h in a water bath at 100 degrees C. The fumigants are collected on a trap composed of Tenax TA and XAD-4 resin, eluted with hexane, and determined by gas chromatography (GC) using electron capture detection or Hall electrolytic conductivity detection. Flame photometric detection in the sulfur mode is used to determine carbon disulfide. Thick-film, wide-bore capillary columns were used exclusively in both the determination and confirmation of the halogenated fumigants. The higher levels of fumigants are also confirmed by full scan GC/mass spectrometry. Samples are analyzed for carbon disulfide, methylene chloride, chloroform, 1,2-dichloroethane, methyl chloroform, carbon tetrachloride, trichloroethylene, 1,2-dibromoethane, and tetrachloroethylene. A total of 25 whole grains, milled grain products, and intermediate grain-based foods analyzed by this method contained fumigant levels up to 51 ppm (carbon tetrachloride in wheat). Recoveries from fortified samples ranged from 82 to 104%. Chromatograms from this purge and trap method are clean, so that low parts per billion and sub-parts per billion levels can be quantitated for the halogenated analytes. The quantitation level for carbon disulfide is 12 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.     

Headspace gas chromatographic method for determination of methyl bromide in food ingredients

A headspace gas chromatographic (GC) method, which can be automated, has been developed for determination of methyl bromide. This method has been applied to wheat, flour, cocoa, and peanuts. Samples to be analyzed are placed in headspace sample vials, water is added, and the vials are sealed with Teflon-lined septa. After an appropriate equilibration time at 32 degrees C, the samples are analyzed within 10 h. A sample of the headspace is withdrawn and analyzed on a gas chromatograph equipped with an electron capture detector (ECD). Methyl bromide levels were quantitated by comparison of peak area with a standard. The standard was generated by adding a known amount of methyl bromide to a portion of the matrix being analyzed and which was known to be methyl bromide free. The detection limit of the method was 0.4 ppb. The coefficient of variation (CV) was 6.5% for wheat, 8.3% for flour, 3.3% for cocoa, and 11.6% for peanuts.     

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)     

Determination of ethyl carbamate in distilled alcoholic beverages by gas chromatography with flame ionization or mass spectrometric detection

Quantitative methods are detailed for determination of ethyl carbamate in distilled alcoholic beverages by capillary gas chromatography with flame ionization detection (GC/FID) and by packed-column gas chromatography/mass spectrometry (GC/MS) using selected ion monitoring. Five g samples of distillate of known ethanol concentration are diluted with water to 25% ethanol (v/v), washed with petroleum ether, and extracted with dichloromethane prior to GC/FID or GC/MS analysis. As necessary, sample extracts that exhibit GC/FID interference are passed through alumina for additional cleanup. When internal standards (tert-butyl carbamate and n-butyl carbamate for GC/FID, or ethyl 13C-15N-carbamate for GC/MS) were used for quantitation, the limit of detection for ethyl carbamate was in the range of 5-25 ppb. Coefficients of variation ranged from 3.5 to 6.0% for GC/FID determinations, and from 1.4 to 3.2% for GC/MS. Correlation between methods for 22 random distillate samples ranging in concentration from approximately 40 to 800 ppb gave a correlation coefficient (r) of 0.996.     

Reduction of Aflatoxin and Fumonisin Contamination in Yellow Corn by High‐Speed Dual‐Wavelength Sorting

A high‐speed dual‐wavelength sorter was tested for removing corn contaminated in the field with aflatoxin and fumonisin. To achieve accurate sorting, single kernel reflectance spectra (500–1,700 nm) were analyzed to select the optimal pair of optical filters to detect mycotoxin‐contaminated corn during high‐speed sorting. A routine, based on discriminant analysis, was developed to select the two absorbance bands in the spectra that would give the greatest classification accuracy. In a laboratory setting, and with the kernels stationary, absorbances at 750 and 1,200 nm could correctly identify >99% of the kernels as aflatoxin‐contaminated (>100 ppb) or uncontaminated. A high‐speed sorter was tested using the selected filter pair for corn samples inoculated with Aspergillus flavus; naturally infested corn grown in central Illinois; and naturally infested, commercially grown and harvested corn from eastern Kansas (2002 harvest). For the Kansas corn, the sorter was able to reduce aflatoxin levels by 81% from an initial average of 53 ppb, while fumonisin levels in the same grain samples were reduced an average of 85% from an initial level of 17 ppm. Similar reductions in mycotoxin levels were observed after high‐speed sorting of A. flavus inoculated and naturally mold‐infested corn grown in Illinois.     

Rapid screening method for detection of deoxynivalenol

A rapid method for detecting deoxynivalenol (DON), also known as vomitoxin, was developed. DON was extracted from grains and other samples with acetonitrile-4% potassium chloride solution (9 + 1). Impurities that would interfere with detection were removed on a C18 silica gel reverse phase column. Water was removed from eluates on a hydrophilic matrix column. DON was detected by thin layer chromatography using an aluminum chloride solution to develop the blue response characteristic of the mycotoxin. Total time involved is approximately 30 min. The method was applicable to corn, wheat, and barley at detection levels of 1 ppm, and oats at 1.5 ppm. It is applicable to environmental samples (soil, green plants, and water) at detection levels of 0.75 ppm.     

Comparison of methodology for determination of ethylene dibromide in grains and grain-based foods

Three commonly used methods for determination of ethylene dibromide (EDB) in grains and grain products have been compared. EDB residues were extracted by soaking in hexane, triple co-distillation with hexane from an aqueous sample solution, and soaking in acetone-water (5 + 1). Each method was used for triplicate analyses of 12 samples containing incurred residues of EDB ranging from about 10 to 1000 ppb and representing whole grains (wheat and oats) and intermediate grain-based products such as corn meal and flour. The 4-day hexane soaking method extracted the least EDB. In some cases, this was half of the amount determined by the other methods. Levels from soaking in acetone-water were equal to, or up to 25% greater than, those from distillation. Although soaking for 2 days is required for whole grains in the method, a period of only 16 h was found acceptable for ground products. Results were obtained faster with the distillation method, but more analyst time per sample was required. A single distillation recovered about 80% (40-60% from wheat) of total EDB extracted by triple distillation. Foaming was reduced by the addition of concentrated H2SO4 to the aqueous hexane-sample mixture, plus stirring during distillation, thereby allowing complete recovery of the hexane.     

Determination of ethylene dibromide in foods and grains by high resolution capillary gas chromatography with electron capture detection

Ethylene dibromide (EDB) levels in food samples were determined by gas chromatography with a high-resolution capillary column and electron capture detector. The capillary column used was 3 mm id X 25 m cross-linked 5% phenylmethyl silicone. Column temperature was set at 40 degrees C by a coolant containing carbon dioxide gas. Optimum temperatures of the injection port and detector were 200 and 350 degrees C, respectively. The detection limit was 0.5 ppb and linear from 1 to 20 pg on the dynamic range. EDB residues in food samples were extracted with n-hexane by steam distillation. A few impurity peaks appeared near EDB on the chromatogram; however, the EDB peak was resolved. Recoveries of EDB from wheat and brown rice ranged from 66.1 to 99.6%. EDB was detected in 3 samples of imported wheat at a range of 0.74-1.70 ppb, and was not detected at all in 37 samples. The EDB remaining in EDB-fortified cookies after baking was examined. The amounts of EDB were reduced to 30 to 50% of the original amounts by kneading the dough, and to below 1.5% by baking.     

Volatile organic compounds in foods: a five year study

A purge and trap procedure was used with gas chromatography-mass spectrometry determination to analyze 70 foods for volatile organic compounds (VOCs). The results from analyses over a 5 year period (1996-2000) are reported. VOCs were found in at least one sample of all foods tested, although no single compound was found in each of the foods. The total amount of VOCs found in a single food item over the 5 year period ranged from 24 to 5328 ppb, with creamed corn (canned) the lowest and cheddar cheese the highest. Benzene was found in all foods except American cheese and vanilla ice cream. Benzene levels ranged from 1 to 190 ppb, with the highest level found in fully cooked ground beef. Benzene was found in 12 samples of cooked ground beef, with an average of 40 ppb. Benzene levels above 100 ppb were also seen in at least one sample each of a cola (138 ppb), raw bananas (132 ppb), and cole slaw (102 ppb). This compares to a maximum contaminant level of 5 ppb set by the U.S. EPA for drinking water.     

Determination of methyl bromide in foods by headspace capillary gas chromatography with electron capture detection

Methyl bromide (MB, bromomethane) is determined in a variety of foods by headspace capillary gas chromatography with electron capture detection. The comminuted food sample as an aqueous sodium sulfate slurry is equilibrated with stirring for 1 h at room temperature before a 1 mL headspace aliquot is removed and injected using a modified on-column syringe needle. Methyl bromide is cryogenically focussed at -60 degrees C and then eluted by temperature programming. The procedure requires blending of soft samples, e.g. raisins, prunes, or oranges, and ultrasonic homogenization of hard samples, e.g. wheat, cocoa beans, corn, or nuts, with portions of water and ice so the final temperature of the food-water slurry is less than 1 degree C. A 20 g aliquot (4 g food) is then added to a cold headspace vial containing 4 g sodium sulfate. Losses of MB during a 3.5 min ultrasonic homogenization of wheat were 11% at 0.95 ppb and 4.4% at 4.8 ppb. For flour, cocoa, and finely divided spices, which do not require blending, 4 g is added to the cold headspace vial containing 16 mL cold water and 4 g sodium sulfate. Studies show that comminution of wheat or peanuts must be carried out to release MB trapped within the food so the headspace equilibrium can be attained in 1 h as well as to obtain homogeneous samples and representative sampling. No interferences were noted with the above foods or with many grain-based baking mixes analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of cytotoxic trichothecenes in corn by cell culture toxicity assay

The great sensitivity of some cell species to toxins has been adapted to a direct biological determination of trichothecene contamination of food and feeds. The murine spleen lymphocyte stimulated by PHA (Phaseolus vulgaris phytohaemagglutinin) appeared to be the most convenient cells because of their particular sensitivity to cytotoxic trichothecenes and the opportunity to translate this cytotoxicity to immunosuppressive hazard, one of the most important concerns for trichothecenes. In this paper, the use of cell cultures was adapted for a survey of corn. The toxins were extracted by aqueous methanol, and the extract was defatted with hexane and purified on a silica gel/Florisil column. This extract was then used for a gas chromatographic (GC) determination and the biological test. The growth of cells was measured by the incorporation of tritiated thymidine (3H Tdr), and the inhibition was expressed by the IC50: concentration of corn extract inhibiting by half the 3H Tdr incorporation. We have tested pure toxins, control corn, corn spiked with T-2 toxin, corn experimentally inoculated with toxigenic Fusarium strains, and naturally contaminated corn. A good correlation exists between IC50 and the T-2 toxin concentration as determined by GC analysis. The response is not affected by the presence of zearalenone or by small amounts of deoxynivalenol. A quantitative evaluation of cytotoxic trichothecenes in corn is valuable in the range of 100 ppb to 10 ppm, expressed as T-2 toxin equivalents. The result is obtained in 48 h.