Melamine has been used for the adulteration of cereal flours in order to increase their apparent protein content. Crude melamine may contain several by-products, i.e. ammeline, ammelide, and cyanuric acid. The simultaneous analysis of all four chemicals is difficult because of the formation of an insoluble salt between melamine and cyanuric acid. A simple and convenient high-performance liquid chromatography (HPLC) method for the detection of the adulteration of cereal flours with all four chemicals is proposed herein. The precipitate formation between melamine and cyanuric acid was prevented by using alkaline conditions (pH 11-12) for both standards preparation and sample extraction. The method uses matrix-matching, which involves the construction of a calibration curve on a blank (negative control) matrix, which is then used for the quantitation of melamine and by-products in adulterated (positive) samples. Matrix-matching compensates for analyte losses during sample preparation, and for matrix effects. The method was successfully applied to wheat, corn, and rice flours, and is expected to be applicable (with some modifications) to soy flour as well. The method allows for the detection of melamine, ammeline, and ammelide at approximately 5 microg g(-1), and cyanuric acid at approximately 90 microg g(-1) in wheat flour.

The effects of cotton-corn rotation and glyphosate use on levels of soil-borne Aspergillus flavus, aflatoxin and fumonisin contamination in corn and cotton seed were determined during 2002-2005 in Stoneville, Mississippi (USA). There were four rotation systems (continuous cotton, continuous corn, cotton-corn and corn-cotton) for both glyphosate-resistant (GR) and non-GR cultivars-herbicide system arranged in a randomized complete block design with four replications. Aspergillus flavus populations in surface (5-cm depth) soil, sampled before planting (March/April), mid-season June) and after harvest (September), ranged from 1.47 to 2.99 log (10) cfu g(-1) soil in the four rotation systems. Propagules of A. flavus were higher in the continuous corn system compared to the continuous cotton system on three sample dates, and cotton rotated with corn decreased A. flavus propagules in three of nine sample dates. Propagules of A. flavus were significantly greater in plots with GR cultivars compared to non-GR cultivars in three samples. In cotton seed, aflatoxin and fumonisin levels were similar (< or = 4 microg kg(-1) and non-detectable, respectively) regardless of rotation and glyphosate. In corn grain, aflatoxin was above the regulatory level (> or = 20 microg kg(-1)) only in GR cultivar in 2004 and 2005. Fumonisin was higher in non-GR cultivar (4 mg kg(-1)) regardless of rotation in 2004; however, in 2002, 2003 and 2005, aflatoxin and fumonisin levels were similar regardless of rotation and glyphosate. These results indicate the potential for increased aflatoxin and fumonisin levels (1 of 4 years) in corn; however, climatic conditions encountered during this study did not allow for mycotoxin production. In laboratory incubation studies, fairly high concentrations of glyphosate were required to inhibit A. flavus growth; however no short-term effect of soil treatment with glyphosate on A. flavus populations were observed. These data suggest that altered populations of A. flavus or higher aflatoxin concentrations in corn grain were due to indirect effects of the GR cropping system.

The commercial processing of oats is different from that of other cereals, such as wheat and maize. In northwest Europe, oats also appear to be more susceptible to contamination with HT-2 and T-2 toxins than other cereals. Mycotoxins, such as deoxynivanol and zearalenone, in cereals are already controlled by EU legislation. With regard to additional, impending legislation, this study examined HT-2 and T-2 toxins together with zearalenone, deoxynivalenol and other related toxins in a commercial oat mill and how the concentrations varied from raw oats to the final prepared oat flakes. Concentrations of each Fusarium mycotoxin fell by 90-95% during the process, with the major loss being a physical distribution occurring at the de-hulling stage. Initial studies of losses occurring at other stages, such as kilning or de-branning of prepared oat groats, suggest these to be small. The use of colour sorting after kilning showed higher concentrations of each mycotoxin in the discoloured groats. The feasibility of developing a predictive tool for the oat industry is examined.

The investigation of adverse health effects associated with fungal mycotoxins requires the measurement of human exposure. Most frequently, this exposure is estimated from contamination levels of raw foodstuffs, which are the primary source of toxin exposure, and data on food consumption patterns. However, variations in food preparation methods, food intake, contamination level, intestinal absorption, toxin distribution and excretion lead to individual variations in toxin exposure that are more readily measured with a biomarker. Fumonisin biomarkers have been sought in the measurement of levels of the toxin in physiological samples such as serum, urine, faeces, hair and nails. However, due to the low bioavailability of fumonisin, these samples pose a variety of analytical challenges and also still require validation as biomarkers. The most widely researched fumonisin biomarkers have been those related to the disruption of de novo sphingolipid biosynthesis, namely elevated levels of the sphingoid base, sphinganine, or of its ratio with sphingosine. Elevation of these parameters in humans would potentially provide a biomarker of biochemical effect. A number of investigations into the possible elevation of sphinganine (or its ratio with sphingosine) in human blood and urine have generally failed to correlate with estimates of fumonisin exposure. The sphingoid bases occur naturally in human blood and urine such that their levels have normal ranges, which can be influenced by dietary factors other than fumonisin ingestion. The lower exposures from human diets, as compared with doses in experimental animals, have made detection of changes in these sphingoid biomarkers problematic.

A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in crude grain extracts without clean-up has been applied to the analysis of spelt, rice and barley. Method performance characteristics were determined after spiking blank samples at multiple levels and were found to be comparable for all investigated matrices as regards linearity (linear calibration functions were obtained for all analyte/matrix combinations except for moniliformin), precision (coefficient of variations <6%) and sensitivity. Matrix-induced signal suppression/enhancement was studied in detail and varied significantly between the investigated matrices, as well as between individual samples (relative standard deviation was as high as 40% within three rice varieties) and individual toxins. It was concluded that a reliable quantitative analysis using matrix-matched calibration requires careful consideration of the model matrix, which should match the investigated samples as close as possible.

This review focuses on recent developments in immunochemical methods for detection of mycotoxins, with a particular emphasis on simultaneous multiple analyte determination. This includes high-throughput instrumental analysis for the laboratory environment (microtitre plate enzyme-linked immunoabsorbant assay (ELISA), different kinds of immunosensors, fluorescence polarization immunoassay, and capillary electrophoretic immunoassay), as well as rapid visual tests for on-site testing (lateral-flow, dipstick, flow-through and column tests). For each type of immunoassay, perspectives for multiple analyte application are discussed and examples cited.

There has been interest in the development of techniques for the rapid early detection of mycotoxigenic moulds in the food production chain. The development of sensor arrays that respond to the presence of different volatiles produced by such moulds has been examined as a potential method for the development of such detection systems. Commercial devices based on such sensor arrays, so-called 'electronic noses', have been examined extensively for the potential application of determining the presence of mycotoxigenic moulds in food raw materials. There is also interest in using the qualitative volatile production patterns to discriminate between non-mycotoxigenic and mycotoxigenic strains of specific mycotoxigenic species, e.g. Fusarium section Liseola, Penicillium verrucosum and Aspergillus section Nigri. This paper reviews the technology and available evidence that the non-destructive analysis of the headspace of samples of food raw materials or the discrimination between strains (mycotoxigenic and non-mycotoxigenic) can be determined using volatile fingerprints.

Aspergillus niger and A. tubingensis, species belonging to section Nigri, are commonly found in plant products and processed food, such as grapes, cereals, coffee, and derived products. These two species are very difficult to differentiate by classical morphological criteria and some isolates are known to produce ochratoxin A. The exact identification of these two species is very important to avoid the overestimation of toxicological contamination and related risks. A polymerase chain reaction (PCR)-based identification and detection assay was developed as a tool to identify A. niger and A. tubingensis, using molecular differences obtained by sequencing the calmodulin gene. Two pairs of species-specific primers were designed and empirically evaluated for PCR identification of A. niger and A. tubingensis. Species-specific PCR products generated by each primer set were 505 bp (A. tubingensis) and 245 bp (A. niger) in length, which could be potentially useful for a multiplex PCR assay. The sensitivity of this assay was about 10 pg DNA in a 25-microl PCR reaction volume, using pure total DNA of the two species. The method described in this study represents a rapid and reliable procedure to assess the presence in food products of two ochratoxigenic species of section Nigri.

Single-stranded conformational polymorphism (SSCP) analysis for genetic diversity studies has been widely applied to detect indirectly sequence differences up to a single base in amplified DNA fragments of the same length, representing an alternative to gene sequencing. In this study SSCP analysis was used to detect sequence variations contained in an about 180-bp region of the calmodulin gene in order to identify Aspergillus section Nigri species. The method described shows that fluorescence-based SSCP analysis by capillary electrophoresis is cheaper and faster than direct sequencing, and suitable for computer-assisted analyses allowing discrimination between the Aspergillus species belonging to the Nigri section: A. aculeatus, Aspergillus 'atypic uniseriate', A. brasiliensis, A. carbonarius, A. ellipticus, A. foetidus, A. heteromorphus, A. ibericus, A. japonicus, A. niger, and A. tubingensis.

Black aspergilli, and particularly Aspergillus carbonarius, are responsible for ochratoxin A production in grapes. Correct identification of these species is essential for toxicological risk assessment in grape and wine. A low-complexity oligonucleotide microarray (OLISA, Apibio, F) based on DNA oligonucleotides probes, obtained from sequences of the calmodulin gene, was set up in order to detect A. carbonarius, A. japonicus/A. aculeatus and A. ibericus isolated from grape. The designed microarray distinguished all Aspergillus species and the detection limit for A. carbonarius was 3.2 pg of DNA as a template for the PCR reaction. This microarray offers a quick and parallel analysis to detect individual Aspergillus species in pure cultures and in naturally contaminated grape samples.

Fusarium proliferatum is a major cause of maize ear rot and fumonisin contamination and also can cause wheat kernel black point disease. The primary objective of this study was to determine whether nine F. proliferatum strains from wheat from Nepal can cause black point and fumonisin contamination in wheat kernels. For comparison, the study included three Fusarium strains from US maize. In test 1, all the strains but one produced significant symptoms of kernel black point; two strains decreased kernel yield; and four strains contaminated kernels with fumonisins B(1), B(2) and B(3) as determined by liquid chromatography-mass spectroscopy. Strain Ggm202 from Nepal, which produced the highest levels of fumonisins (mean = 49 microg g(-1)) on five wheat cultivars in test 1, was confirmed to produce fumonisins (mean = 38 microg g(-1)) on two cultivars in test 2. The data indicate a potential for fumonisin contamination of wheat infected with F. proliferatum.

The accuracy of a quantitative polymerase chain reaction assay in quantifying the DNA of trichothecene-producing F. culmorum and F. graminearum within harvested wheat grains and head tissue was evaluated in comparison with incidences of infected kernels and deoxynivalenol levels. In a first experiment, six durum and bread wheat varieties were grown in randomized plots for a 2-year period, and inoculated with Fusarium macroconidia at six growth stages between heading and dough ripening, to obtain a wide range of Fusarium head blight incidences. There was a close relationship between fungal DNA and the amount of deoxynivalenol, and this relationship was consistent over Fusarium species, wheat species and varieties, and over a wide range of Fusarium head blight infection. In a second experiment potted wheat plants were grown under environmentally controlled conditions and inoculated with the two Fusarium species at full flowering; head samples were collected before inoculation and after 6 h to 12 days, and processed by the quantitative polymerase chain reaction assay. This assay made it possible to detect the dynamic of fungal invasion in planta after infection had occurred, and to single out the presence of infection before the onset of the disease symptoms: A robust detection of the infection occurred within 18-24 h for F. culmorum, and within 2-9 days for F. graminearum.