A. De Girolamo

Rapid and non-invasive analysis of deoxynivalenol in durum and common wheat by Fourier-Transform Near Infrared (FT-NIR) spectroscopy

Fourier transform near-infrared spectroscopy (FT-NIR) was used for rapid and non-invasive analysis of deoxynivalenol (DON) in durum and common wheat. The relevance of using ground wheat samples with a homogeneous particle size distribution to minimize measurement variations and avoid DON segregation among particles of different sizes was established. Calibration models for durum wheat, common wheat and durum + common wheat samples, with particle size <500 µm, were obtained by using partial least squares (PLS) regression with an external validation technique. Values of root mean square error of prediction (RMSEP, 306–379 µg kg–1) were comparable and not too far from values of root mean square error of cross-validation (RMSECV, 470–555 µg kg–1). Coefficients of determination (r 2) indicated an “approximate to good” level of prediction of the DON content by FT-NIR spectroscopy in the PLS calibration models (r 2 = 0.71–0.83), and a “good” discrimination between low and high DON contents in the PLS validation models (r 2 = 0.58–0.63). A “limited to good” practical utility of the models was ascertained by range error ratio (RER) values higher than 6. A qualitative model, based on 197 calibration samples, was developed to discriminate between blank and naturally contaminated wheat samples by setting a cut-off at 300 µg kg–1 DON to separate the two classes. The model correctly classified 69% of the 65 validation samples with most misclassified samples (16 of 20) showing DON contamination levels quite close to the cut-off level. These findings suggest that FT-NIR analysis is suitable for the determination of DON in unprocessed wheat at levels far below the maximum permitted limits set by the European Commission.

Comparison of urinary sphingolipids in human populations with high and low maize consumption as a possible biomarker of fumonisin dietary exposure.

The increased sphinganine/sphingosine (SA/SO) ratio has previously been shown as a biomarker of fumonisin exposure in experimental animals and has been proposed as a tool to assess human exposure to fumonisin mainly occurring through the dietary consumption of fumonisin contaminated maize-based foods. Sphinganine and sphingosine were measured in urines of humans resident in two areas of North Argentina and South Brazil with high maize consumption and compared with urine samples collected in areas with very low or no maize consumption, such as Central Argentina and Southern Italy. The pattern of SA/SO values in the two groups with no maize consumption (assumed as controls) was similar, with all SA/SO values lower than one. Mean SA/SO ratio was 1.27 in urine of subjects with high maize consumption (n = 123) and 0.36 in controls (n = 66) and the difference was statistically significant (p<0.001). The mean fumonisin level in maize samples collected in North Argentina and South Brazil was 0.35 mg kg−1 (n = 40). Although a similar maize and fumonisin intake was recorded for the two groups of populations, the mean SA/SO ratio in South Brazil (1.57) was significantly higher (p<0.05) than that of North Argentina (0.69). These data suggest that the higher SA/SO values observed in South Brazil cannot be associated with high fumonisin exposure and further studies are necessary to provide convincing evidence for using the SA/SO ratio as a biomarker of human fumonisin exposure.

Toxigenic profile of Alternaria alternata and Alternaria radicina occurring on umbelliferous plants

Alternaria alternata and Alternaria radicina are fungal species that occur in several food crops and may produce mycotoxins and phytotoxins. The toxigenic profile of A. alternata and A. radicina isolated from carrot and other umbelliferous plants was determined by growing the fungus on rice and carrot discs. Most of the tested isolates of A. alternata produced the mycotoxins tenuazonic acid, alternariol, alternariol methyl ether and altertoxin–I on rice. Only alternariol and alternariol methyl ether were produced on carrot discs. When cultured on rice, none of the isolates of A. alternata from umbelliferous plants produced AAL toxins and fumonisins. AAL toxins, but not fumonisins, were instead produced by A. alternata f. sp. lycopersici isolate NRRL 18822 isolated from tomato. A. radicina produced the phytotoxic compounds radicinin, epi–radicinol and radicinol on carrot discs, whereas it produced radicinin and radicinol on rice. Although A. alternata has been frequently found in organic carrots, none of the above mycotoxins was detected in carrot roots or in carrot commercial products. The reduction of alternariol and alternariol methyl ether during carrot juice processing at laboratory scale was estimated to be >98%. Based on these findings and previous reports, it can be concluded that Alternaria mycotoxins in carrots do not represent a hazard for consumers.

Determination of Fumonisins B1 and B2 in Maize-Based Baby Food Products by HPLC with Fluorimetric Detection after Immunoaffinity Column Clean-up

A method for the determination of fumonisin B1 (FB1) and B2 (FB2) in different commercial maize-based products for infants and young children was developed and tested in a limited validation study involving 3 laboratories. The method used extraction at 55 °C with an acidic mixture of methanol-acetonitrile-phosphate/citrate buffer, clean-up through immunoaffinity column and fumonisin determination by high performance liquid chromatography with automated pre-column derivatisation with o-phthaldialdehyde. Recovery experiments were performed at five spiking levels in the ranges of 80-800 µg/kg FB1 and 20-200 µg/kg FB2. Mean recoveries ranged from 83 to 97% for FB1 and from 61 to 78% for FB2. Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 5 to 12% for FB1 and from 8 to 13% for FB2, whereas relative standard deviation for between-laboratory reproducibility (RSDR) ranged from 6 to 10% for FB1 and from 9 to 16% for FB2. The limit of quantification of the method (signal to nois...

Results of a proficiency test for multi-mycotoxin determination in maize by using methods based on LC-MS/(MS)

Liquid chromatography coupled with single or tandem mass spectrometry (LC-MS/(MS)) is routinely used for the simultaneous determination of mycotoxins in food and feed although official methods using this technique have not yet been adopted by the European Committee for Standardization and the Association of Analytical Communities. A proficiency test (PT) was conducted for the simultaneous determination of up to 11 mycotoxins (aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), aflatoxin G 2 (AFG 2 ), ochratoxin A (OTA), deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEA), fumonisin B 1 (FB 1 ) and fumonisin B 2 (FB 2 )) in maize using LC-MS/(MS) to benchmark laboratories currently using this technique and to obtain information on currently used methodologies and method-related performances. Each participant received the following: instructions; a comprehensive questionnaire; a mixed mycotoxins calibration solution; a spiking solution (AFB 1 , AFB 2 , AFG 1 and AFG 2 , OTA, DON, T-2, HT-2, ZEA, FB 1 and FB 2 ); and two test materials, namely a contaminated maize sample and a blank maize sample to be spiked with a spiking solution containing 11 mycotoxins. Laboratory results were rated with z-scores. Of the 64 laboratories enrolled in the PT, 41 laboratories from 14 countries returned 43 sets of results for various combinations of analytes. The majority of laboratories (61%) reported results for all 11 mycotoxins, whereas the remaining laboratories reported results for a restricted combination (from 2 to 10 analytes). For contaminated maize and spiked maize the percentage of satisfactory z-score values (|z| ≤2) were: DON 55% and 49%, FB 1 50% and 30%, FB 2 52% and 38%, ZEA 68% and 64%, T-2+HT-2 toxins 82% and 85%, OTA 58% and 60%, AFB 1 56% and 62%, AFG 1 73% and 84%, AFB 2 40% and 78%, AFG 2 64% and 78%, respectively. The poorest performance (|z| >3) was obtained for FB 1 (31%), FB 2 (32%), AFB 1 (32%) and AFB 2 (32%) in contaminated maize and for DON (35%), FB 1 (63%) and FB 2 (52%) in spiked maize. Mean recovery results were acceptable for all mycotoxins (74% to 109%), except for fumonisins, where these were unacceptably high (159% for FB 1 and 163% for FB 2 ). A robust and reliable method for simultaneous determination of 11 mycotoxins in maize could not be identified from the results of this PT. Additional experimental work is necessary to set up a method suitable for inter-laboratory validation. The results of this PT and the relevant method’s details can be useful to identify methodology strengths and weaknesses.

Comparison of methods and optimisation of the analysis of fumonisins B1 and B2 in masa flour, an alkaline cooked corn product

A comparison study of different extraction and clean-up procedures for the liquid chromatographic analysis of fumonisins B1 (FB1) and B2 (FB2) in corn masa flour was performed. The procedures included extraction (heat or room temperature) with acidic conditions or EDTA-containing solvents, and clean-up by immunoaffinity or C18 solid-phase extraction columns. Thereafter an analytical method was optimised using extraction with an acidic mixture of methanol–acetonitrile–citrate/phosphate buffer, clean-up through the immunoaffinity column and determination of fumonisins by liquid chromatography with automated pre-column derivatisation with o-phthaldialdehyde reagent. Recovery experiments performed on yellow, white and blue masa flours at spiking levels of 400, 800 and 1200 µg kg−1 FB1 and of 100, 200 and 300 µg kg−1 FB2 gave overall mean recoveries of 99% (±6%) for FB1 and 88% (±6%) for FB2. Good recoveries (higher than 90% for both FB1 and FB2) were also obtained with corn tortilla chips. The limits of quantification of the method (signal-to-noise ratio of 10) were 25 µg kg−1 for FB1 and 17 µg kg−1 for FB2. The method was tested on different commercial corn masa flours as well as on white and yellow corn tortilla chips, showing fumonisin contamination levels (FB1 + FB2) up to 1800 µg kg−1 (FB1 + FB2) in masa flour and 960 µg kg−1 in tortilla chips. Over 30% of masa flours originating from Mexico exceeded the European Union maximum permitted level.

Effect of alkaline cooking of maize on the content of fumonisins B1 and B2 and their hydrolysed forms.

The effect of nixtamalization on the content of fumonisins (FBs), hydrolysed (HFBs) and partially hydrolysed (PHFBs) fumonisins in maize was investigated at laboratory-scale. Maize naturally contaminated with FBs and PHFBs was cooked with lime. Starting raw maize, steeping and washing waters and final masa fractions were analysed for toxin content. Control-cooking experiments without lime were also carried out. The nixtamalization reduced the amount of FBs and PHFBs in masa and converted them to HFBs. However, the three forms of fumonisins collected in all fractions amounted to 183%, indicating that nixtamalization made available forms of matrix-associated fumonisins that were then converted to their hydrolysed forms. Control-cooking enhanced FBs and PHFBs reduction, due to the solubility of fumonisins in water during the steeping process, but did not form HFBs. These findings indicate that benefits associated with enhancing the nutritional value of nixtamalized maize are also associated with a safer product in terms of fumonisin contamination.

Critical evaluation of LC-MS-based methods for simultaneous determination of deoxynivalenol, ochratoxin A, zearalenone, aflatoxins, fumonisins and T-2/HT-2 toxins in maize

The results of a proficiency test for the LC-MS/(MS) determination of up to 11 mycotoxins (aflatoxins B1, B2, G1 and G2, fumonisins B1 and B2, ochratoxin A, deoxynivalenol, T-2 and HT-2 toxins and zearalenone) in maize were evaluated to identify possible strengths and weaknesses of various methodologies used by the 41 participating laboratories. The majority of laboratories (56%) used mixtures of acetonitrile:water for extraction. Other laboratories used methanol:water mixtures (17%) or performed two consecutive extractions with phosphate buffer solution (PBS) followed by methanol (15%). Few laboratories used mixtures of acetonitrile:water:methanol (7%), water:ethyl acetate (2.5%) or PBS alone (2.5%). The majority of laboratories (58%) used a clean-up step prior to chromatography. The remaining laboratories analysed crude extracts (37%) or used a mixed approach (5%). The amount of sample equivalent injected into LC-MS/(MS) ranged between 0.1-303 mg for purified extracts and 0.08-20 mg for directly analyse...

Influence on functional parameters of intestinal tract induced by short-term exposure to fumonisins contaminated corn chyme samples

The gut is a possible target toward mycotoxin fumonisins (FBs) exposure. The study aims to investigate the effects induced by FBs contaminated-corn chyme samples on functional parameters of human and rat intestine by using Ussing chamber. Fumonisins-contaminated corn and processed corn samples were undergone to in vitro digestion process and then added to luminal side. A reduction (about 90%) of short circuit current (Isc μA/cm(2)) during exposure of human colon tissues to fumonisins-free corn chyme samples was observed, probably related to increased chyme osmolality. This hyperosmotic stress could drain water towards the luminal compartment, modifying Na(+) and Cl(-) transports. The presence of FBs in corn chyme samples, independently to their concentration, did not affect significantly the Isc, probably related to their interference towards epithelial Na(+) transport, as assessed by using a specific inhibitor (Amiloride). The rat colon tract represents a more accessible model to study FBs toxicity showing a similar functional response to human. In the rat small intestine a significant reduction (about 15%) of Isc parameter during exposure to uncontaminated or FBs contaminated corn chyme samples was observed; therefore such model was not suitable to assess the FBs toxicity, probably because the prevalent glucose and amino acids electrogenic absorption overwhelmed the FBs influence on ionic transport.

Dose-dependent lipid peroxidation induction on ex vivo intestine tracts exposed to chyme samples from fumonisins contaminated corn samples.

Fumonisins (FBs), Fusarium mycotoxins common food contaminant, are a potent inducer of oxidative stress and lipid peroxidation in intestinal cells. In order to verify this toxic effect in intestine tract, the aim was to assess lipid peroxidation (as malondialdehyde MDA increased levels) on intestine rat samples exposed to chyme samples from in vitro digestion of FBs contaminated corn samples. Naturally (9.61±3.2 μg/gr), artificially (726±94 μg/gr) and spiked corn samples at EU permitted FBs levels were digested and added to luminal side of Ussing chamber for 120 min. Fumonisins-free corn sample was used as control. The MDA increase was observed just in 83% of intestine samples exposed at EU FBs levels and the digestion process seems to reduce this incidence (50% of samples). Malondialdehyde levels were FBs dose- and subject-related and ranged from 0.07±0.01 to 3.59±0.6 nmol/mg. Highest incidence and MDA % increment (I) were found when intestine tracts were exposed to chymes from artificially corn sample. The induction of lipid peroxidation induced by FBs could be due to interactions between FBs and intestinal membranes, with consequent modifications in membrane permeability and oxygen diffusion-concentration, as suggested by other authors.