Minicolumn detection methods for aflatoxin in yellow corn: collaborative study

The CPC modified method, the Holaday modified method, and the combination of the 2 procedures have been compared. The CPC modified method involves more cleanup steps but has a more sensitive column. The Holaday modified procedure has fewer cleanup steps, but the column is more difficult to interpret. The combination CPC-Holaday, which has proven to be the most satisfactory, combines the speed and simplicity of the Holaday extraction and the sensitivity of the Velasco minicolumn used in the CPC method. Levels of 10 ng/g were detected by 89% of the collaborating laboratories using the combination, Holaday-Velasco, method. The combination method has been adopted as official first action.     

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.     

Modification of the rapid screening method for aflatoxin in corn for quantitative use

A study was made to determine if the official AOAC method for screening of aflatoxin in corn could be modified for use as a quantitative method. Several different corn products were analyzed using the modified method, with an average savings of over 1 h/sample vs the CB method. Average recoveries for aflatoxin B1 were 94% for the low level spiked samples and 108% for the high level. Samples of corn and corn products containing naturally incurred aflatoxin were also analyzed with the modified method, and the results compared favorably with those obtained by the CB method.     

Simple method for simultaneous detection of aflatoxin and zearalenone in corn.

A method is described for the simultaneous detection of alfatoxin and zearalenone in corn at 5 and 200 ppb, respectively. No evaporation of solvent is required and the procedure is simple enough to be considered for use at marketing locations. The presence of absence of these myocotoxins can be determined in 10-20 min/sample. The procedure involves an initial blender extraction with methanol, partitioning of fat and pigments into 1-1,2-trichlorotrifluoroethane (Freon-113) from an aqueous ammonium sulfate layer, followed by extraction of aflatoxin from the aqueous layer with chlorobenzene. The chlorobenzene extract can be spotted directly onto a thin layer chromatographic plate which requires only 4 min development. Concentrations of aflatoxin and zearalenone can be estimated by visual comparison of sample spots with standards.     

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.     

Laser fluorometric determination of aflatoxin B1 in corn.

A 2-step chromatographic separation, using both thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC), in conjunction with the high sensitivity of laser fluorometry permits extension of the detection limits of aflatoxin contamination in corn to 0.1 ppb (microgram/kg) with a 26% root mean square variation. Aflatoxin B1 is extracted from corn with water-methanol and cleaned up by TLC. The recovery of aflatoxin from the TLC plates was linear from 10 to 1000 pg. Aflatoxin B1 is converted to the more highly fluorescent B2A derivative by treatment with 1N HCl. Experiments with aflatoxin B1 standard establish a constant conversion to B2A over approximately 3 orders of magnitude in B1 concentration. An extract of the B2A aflatoxin derivative is injected onto a reverse phase HPLC column. A flowing droplet of eluant is irradiated by an amplitude-modulated 325 nm He-Cd ion laser beam, and fluorescence from the droplet is detected by a lock-in amplifier in phase with the laser modulation. Several chromatograms are presented that demonstrate the capability of this procedure for removing interfering components in the corn extract.     

Nitrogen fertilizer influence on aflatoxin contamination of corn in Louisiana

Studies were conducted in 1997 and 1998 on a Gigger silt loam at the Macon Ridge Research Station at Winnsboro, LA, to determine the influence of nitrogen (N) rate, timing, and starter nitrogen fertilizer on aflatoxin contamination in corn. Fertilizer N (0, 50, 100, 150, 200, and 250 lb of N/acre), two timings (at planting and six-leaf stage), and starter N fertilizer (a control and 10 lb of N/acre applied in furrow) were evaluated. Application of starter, N rates, and the interaction of starter with N timing and N rates significantly affected aflatoxin levels. Rates of 50-250 lb of N/acre were 34-43% lower in aflatoxin contamination than plots receiving no N. The application of 10 lb of N/acre starter reduced the aflatoxin levels by 20% compared to the no-starter control.     

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.     

Enzyme-linked immunosorbent assay of aflatoxin B1 in naturally contaminated corn and cottonseed

Naturally contaminated corn and cottonseed samples were screened for aflatoxin B1 (AFB1) by a direct competitive enzyme-linked immunosorbent assay (ELISA). Samples were blended 5 min in an extraction solvent of methanol-water-dimethylformamide (70 + 29 + 1) and filtered. Filtrates were assayed by direct competition between AFB1 in the corn and cottonseed samples and AFB1-peroxidase conjugate for binding to specific antibody adsorbed to a solid phase microtiter plate. Standard curves prepared using the extract of AFB1-free corn and cottonseed samples, and extraction solvent only, showed negligible interference by the sample extract in the performance of ELISA. The AFB1 content in corn and dehulled cottonseed samples as determined by ELISA ranged from 7 to 422 micrograms/kg and 7 to 3,258 micrograms/kg, respectively. When ELISA estimates of AFB1 in corn were compared with values obtained by thin layer chromatography (CB method), the correlation coefficient (n = 10) was 0.95. Average interassay and subsample coefficients of variation for ELISA in corn were 21.4 and 22.0%, respectively. When ELISA estimates of AFB1 in cottonseed were compared with values obtained by liquid chromatography (Pons method), the correlation coefficient (n = 15) was 0.96. Using this ELISA, 36 duplicate sample extracts can be screened for AFB1 in less than 2 h.     

Evaluation of Nutrasweet sludge as a nitrogen fertilizer for corn and wheat

Abstract

NutraSweet sludge, a by‐product of the production of the noncarbohydrate sweetener aspartame, is often used as a N fertilizer for crops. However, its performance with respect to inorganic N fertilizers is not well understood. This work was conducted to compare NutraSweet sludge to ammonium sulfate and urea as an N fertilizer for wheat and corn. Samples from two soils were mixed with one of the three N sources to achieve rates of 0, 25, 50, 100, or 150 mg N kg^‐1. The treated soil was placed in pots, which were used to grow corn or wheat for 45 days in the greenhouse. Above‐ground dry matter yields of com and wheat increased as N rate increased from 0 to 50 or 100 mg N kg^‐1. Above 100 mg N kg^‐1, dry matter yields decreased. In general, at a given N rate, NutraSweet sludge produced dry matter yields that were equal to or higher than those obtained with ammonium sulfate or urea. The results suggest that NutraSweet sludge could be managed as an ammoniacal N fertilizer when applied to crops.     

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.     

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

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

Visual and semiquantitative spectrophotometric ELISA screening method for aflatoxin B1 in corn and peanut products: follow-up collaborative study

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)     

我国东北地区春玉米科学施肥评价

为明确东北农户春玉米施肥现状,实现东北地区春玉米稳产增产和养分高效利用,2019年对东北典型春玉米种植区247个农户的春玉米养分管理调研,基于区域不同产量水平推荐施肥量评价农户施肥量,分析农户施肥存在的问题及其对科学施肥技术的掌握情况.结果表明,调研地区春玉米氮肥(N)、磷肥(P2O5)和钾肥(K2O)的平均施用量分别...     

Evaluation of an attract and kill strategy for western corn rootworm larvae

Western corn rootworm larvae are serious soil dwelling maize pests, and use carbon dioxide (CO₂) to locate maize roots. The efficacy of insecticides can be enhanced by a combination with an attractant used in host finding, known as attract and kill. This study tested the use of CO₂ emitting capsules as an attractant in combination with the soil insecticide tefluthrin. An observation device was developed to study the temporal and spatial distribution changes of the larvae and to test whether these are influenced by the application of the capsules. Furthermore it was evaluated to what extent larvae are killed by the insecticide in combination with the capsules and whether this could be used for an attract and kill strategy to manage this pest.The observation device enabled recovery of 20–40% of the inserted larvae. The spatial analysis of distance indices revealed a sequence of spatial and temporal distribution patterns of the larvae in the root system. This sequence of spatial distribution was disrupted by an application of the capsules around which the larvae started to aggregate. Up to 40% mortality of the larvae with attract and kill was observed and thus could be increased over the conventional application (11% mortality) at lower application rates of tefluthrin. In conclusion an attract and kill strategy might be valuable to target this soil dwelling pest. Experiments under field conditions are needed to explore its potential as a management option for the western corn rootworm. Moreover, a further development of the capsules with host specific cues is needed to increase the attractiveness and subsequent mortality of the larvae.