Free Fumonisins Research Articles

Optimization and Validation of an Analytical Method for the Determination of Free and Hidden Fumonisins in Corn and Corn Products by UHPLC-MS/MS

Fumonisins are mycotoxins and corn has been considered the foodstuff presenting the highest contamination levels by these compounds. In recent years, the presence of hidden fumonisins in corn and corn products has aroused the interest of public health authorities. The aim of this study was to optimize and validate a simple and fast analytical method for the determination of free and hidden fumonisins in corn and corn products by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Several sample treatment methods were assessed for free fumonisins determination, and a method using rapid shaking and sonication, followed by extract dilution without the need for a clean-up step was selected for validation. Hidden fumonisins determination was based on the hydrolyzed forms (HFB1 + HFB2) obtained after sample alkaline hydrolysis. The alkaline hydrolysis efficiency was optimized, with a final selected shaking time of 60 min. Satisfactory recoveries were obtained for the target mycotoxins in the validated method, ranging from 89 to 110%, with relative standard deviations lower than 14%. Linearity was confirmed for the calibration curves at the assessed concentration ranges. The method sensitivity was considered adequate, with limits of quantification ranging from 0.4 μg kg−1 to 17.8 μg kg−1. The proposed analytical method was applied satisfactorily to the analysis of real samples, being considered attractive for routine analyses.

Assessment of Modified Forms of Fumonisins in Corn-Based Products Retailed in Japan by an Alkaline Hydrolysis Method

Fumonisins, which are secondary metabolites produced by some Fusarium species, are detected mainly in corn and corn-based products. Recently, the presence of modified forms of fumonisins in fumonisin-contaminated food products has been reported. In order to evaluate the health risk of modified forms of fumonisins to the Japanese population, we analyzed modified forms of fumonisins in corn-based products retailed in Japan. The modified and free forms of fumonisins in food samples were hydrolyzed by alkaline treatment. The resulting hydrolyzed fumonisins were quantified by LC-MS/MS, and total fumonisins (sum of modified and free forms) was calculated. A total of 166 samples of corn-based products were analyzed over two years. The relative ratios of mean total fumonisins to mean free fumonisins in the cornflakes, corn snacks, corn flour and powdered corn soup samples were 4.7, 2.8, 2.1 and 1.2, respectively. Total fumonisins in the residual solid of five cornflake and three corn snack samples obtained after extraction with methanol-water (3 : 1) were quantified. In the cornflakes and corn snacks samples, 56-72 and 83-98% of the modified forms of fumonisins were present in the residual solid, respectively. The average daily intake of fumonisins from cornflakes and corn snacks by the Japanese population was estimated at 1.1 to 3.9 ng/kg body weight/day when the results of free fumonisins were used for the estimate, but when the results of total fumonisins were used, average daily intake increased about three times and was estimated at 3.3 to 12.5 ng/kg body weigh/day. These results indicate that a risk assessment of fumonisins, including the modified forms of fumonisins, is necessary in order to evaluate the true risk of fumonisins to Japanese people.

Free and hidden fumonisins in Argentinean raw maize samples

The concentrations of free fumonisins (FBs) and hydrolysed fumonisins (HFBs) were determined in 72 maize samples collected in 2017 from five Argentine provinces. The methodology for HFBs analysis consisted of alkaline hydrolysis followed by high performance liquid chromatography with a fluorescence detector (HPLC-FLD). The developed analytical methodology presented percentages of recovery greater than 74%. Limits of detection were 4.5, 13.0 and 12.9 μg/kg for HFB1, HFB2 and HFB3, respectively. Presence of FBs was found in 86% of the samples. In all cases, the concentration of total HFBs (after the hydrolysis treatment) was superior to the free FBs content (HFBs to FBs median ratio of 2.5), which indicates the presence of hidden fumonisins in Argentinean maize. 8% of the traditionally analysed samples exceeded the limit established by the European Commission for FB1 + FB2. When applying alkaline hydrolysis to the samples, 24% of them exceed this limit.

Free and hidden fumonisins in hordei fructus germinatus and their transfer to the decoction

Objective: This study aimed to determine the free and hidden fumonisins in Hordei Fructus Germinatus samples and to investigate the transfer rates of these mycotoxins from Hordei Fructus Germinatus to its decoction. Materials and Methods: The contamination levels of free and hidden fumonisins in a total of 60 Hordei Fructus Germinatus samples were analyzed using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The decoction procedure was simulated with a highly contaminated Hordei Fructus Germinatus sample, and fumonisins in the resulting decoction preparation were determined by LC-MS/MS. Results: Among all the samples, 8.3% were contaminated with free fumonisins (FB1 and/or FB2) and 13.3% were contaminated with total fumonisins (free + hidden, measured as hydrolyzed fumonisins, i.e., HFB1 and/or HFB2). The concentrations of FB1and HFB1reached up to 83 and 95 μg/kg, respectively, whereas FB2and HFB2were detected only in traces. The transfer rates of free and total fumonisins from Hordei Fructus Germinatus to the decoction were 71.8% and 83.3% for FB1and FB2, respectively. In comparison, much lower transfer rates were found for total fumonisins, i.e., 38.2% and 24.7% for HFB1and HFB2, respectively. Conclusion: The incidence and contamination levels of free and hidden fumonisins in Hordei Fructus Germinatus samples were generally low. Regarding decoction preparation, the transfer rates of free fumonisins into the decoction were high, whereas a large part of hidden fumonisins were retained in Hordei Fructus Germinatus rather than migrating into water.

Free and hidden fumonisins in raw maize and maize-based products from China

ABSTRACTThe occurrence of free and hidden fumonisins in raw maize and maize-based products from China was investigated. A total of 58 samples were analyzed using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Among all the samples, 66% were contaminated with free fumonisins above limits of quantitation, and a higher percentage of 86% was found for total fumonisins (free + hidden). The highest contamination levels were observed in dried maize kernels which appeared mouldy, with mean levels of 15,737 and 30,785 µg/kg for free and total fumonisins, respectively. Frozen maize kernels, fresh maize kernels, and maize starch samples exhibited the lowest contamination levels, with no more than 200 µg/kg of total fumonisins. Except for one sample, the concentrations of total fumonisins were greater than those of free fumonisins with all the samples, the ratios of total-to-free fumonisins varied between 1.1 and 5.2, with an average ratio of 2.0.

Effects of pH and Temperature on the Stability of Fumonisins in Maize Products.

This paper is a study of the stability of fumonisins in dough based on maize flour prepared in a phosphate buffer with a pH of 3.5, 5.5 or 7.5 and baked at a temperature within the range of 100–250 °C. Buffers with various pH values were tested, since it is well-known that pH may significantly influence interactions of fumonisins with other substances. A standard analytical procedure was used to determine the concentration of free fumonisins. Hydrolysis in an alkaline medium was then applied to reveal the hidden forms, while the total fumonisins concentations was determined in another measurement. The total concentration of fumonisins was statistically higher in pH = 3.5 and pH = 5.5 than the concentration of free fumonisins; no similar difference was found at pH = 7.5. The applied phosphate buffer pH 7.5 may enhance solubility of fumonisins, which would increase extraction efficiency of free analytes, thereby decreasing the difference between concentrations of total and free fumonisins. Hydrolysed B1 fumonisin (HFB1) and partially hydrolysed B1 fumonisin (isomers a and b: PHFB1a and PHFB1b, respectively) were the main investigated substances. For baking temperatures below 220 °C, fumonisins were slightly more stable for pH = 5.5 than for pH = 3.5 and pH = 7.5. In both of these latter cases, the concentration of partially hydrolysed fumonisins grew initially (up to 200 °C) with an increase in the baking temperature, and then dropped. Similar behaviour was observed for free HFB1, which may suggest the following fumonisin degradation mechanism: initially, the tricarballylic acid (TCA) groups are removed from the molecules, and next, the HFB1 molecules disintegrate.

Comparison of the amount of bioaccessible fumonisin B1 and B2 in maize and rice inoculated with Fusarium verticillioides (MRC 826) and determined by in vitro digestion-preliminary results.

In this study the occurrence of hidden fumonisin B1 (FB1) and fumonisin B2 (FB2) was analysed, on two cereal substrates (maize and rice), inoculated with Fusarium verticillioides (MRC 826), in order to determine the ratio of hidden FB1 and FB2. Two parallel methods were applied: an in vitro human digestion sample pre-treatment and the routine extraction procedure, in both cases with subsequent LC-MS analysis. It was found that all samples showed higher concentration of total fumonisin B1 after digestion, as compared to that of free fumonisin analysed only after extraction. The percentage of the hidden form by maize was 18.8% (±2.4) for FB1 and 36.8% (±3.8) for FB2, while for rice it was 32.3% (±11.3) and 58.0 (±6.8), respectively, expressed as the proportion to total fumonisin B1, for the total dataset. Significant differences were found in the FB1 and FB2 concentration measured after the different digestion phases (saliva, gastric and duodenal) in case of both matrixes. The results are useful for human risk assessment, since both humans and animals may be exposed to markedly higher toxin load, as determined merely by conventional analytical methods.

Open Field Study of Some Zea mays Hybrids, Lipid Compounds and Fumonisins Accumulation.

Lipid molecules are increasingly recognized as signals exchanged by organisms interacting in pathogenic and/or symbiotic ways. Some classes of lipids actively determine the fate of the interactions. Host cuticle/cell wall/membrane components such as sphingolipids and oxylipins may contribute to determining the fate of host–pathogen interactions. In the present field study, we considered the relationship between specific sphingolipids and oxylipins of different hybrids of Zea mays and fumonisin by F. verticillioides, sampling ears at different growth stages from early dough to fully ripe. The amount of total and free fumonisin differed significantly between hybrids and increased significantly with maize ripening. Oxylipins and phytoceramides changed significantly within the hybrids and decreased with kernel maturation, starting from physiological maturity. Although the correlation between fumonisin accumulation and plant lipid profile is certain, the data collected so far cannot define a cause-effect relationship but open up new perspectives. Therefore, the question—“Does fumonisin alter plant lipidome or does plant lipidome modulate fumonisin accumulation?”—is still open.

Fumonisins and their masked forms in maize products

In recent years many papers on masked mycotoxins in maize-based products appeared, including reports on fumonisins capable to form non-covalent bonds with food macro constituents. Such so-called “hidden fumonisins” are frequently present in food at quantities higher than the free forms. The aim of this work was to assess levels of free and total (free + hidden) fumonisins (B1, B2 and B3) in 88 maize products available on the Polish retail market. Isotope dilution ion trap mass spectrometery coupled to a high performance liquid chromatography was used. 57% of all tested samples contained free fumonisins at concentrations above our limit of quantification LOQ (mean 390 ± 676 μg/kg). More than 77% of the samples contained free + hidden fumonisins at concentrations above LOQ (mean 574 ± 1177 μg/kg). The highest mean fumonisins concentrations 1006 ± 1131 μg/kg, 1651 ± 2317 μg/kg, respectively for free and free + hidden forms were observed in the group of maize snacks. The lowest fumonisin concentrations were found in maize-based starch concentrate products. None of the tested products within that group had free fumonisins concentrations above LOQ, while mean concentration of free + hidden fumonisins was as low as 82 ± 42 μg/kg. In thermally processed products like corn flakes and various snacks the hidden-to-free fumonisin concentration ratio was higher than in unprocessed products like flour, groats or raw popcorn grains.

Free and hidden fumonisins in Brazilian raw maize samples

Fumonisins are secondary metabolites produced primarily by fungi strains that belong to the genera Fusarium and Alternaria, which have been shown to be highly prevalent in maize crops. Some authors have documented the presence of hidden forms of fumonisins occurring in raw maize. This purpose of this study was to determine the occurrence of free and hidden fumonisins in raw maize. The concentrations of fumonisins in 72 naturally contaminated maize samples were analyzed using liquid chromatography coupled to mass spectrometry. The performance parameters of the method to determine free fumonisins forms (FB1 and FB2) and hydrolyzed fumonisins forms (HFB1 and HFB2) were evaluated using the standards from the Commission of the European Communities (Commission, 2002). The analytical methods employed fell within the established guidelines. The amount of total fumonisins measured based on the hydrolyzed forms (HFB1 + HFB2) was 1.5–3.8 times greater than the amount of free fumonisins (FB1 + FB2). The concentration of hidden fumonisins was calculated by subtracting the levels of free fumonisins from the total fumonisin levels. The levels of hidden fumonisins were calculated to be 0.5–2.0 times greater than the level of free fumonisins. A strong positive correlation (R = 0.97) was observed between free fumonisins (FB1 + FB2) and. total fumonisins (HFB1 + HFB2). Based on this correlation, a predictive model was generated to estimate the total fumonisin level based on the measured/reported free fumonisin concentration. These results show that the risk of exposure to fumonisins is likely underestimated if only free fumonisins are considered. However, the predictive model could be a novel approach to estimating the total amount of fumonisins in maize samples without needing to perform expensive and time-consuming analytical methods.

Determination of the amount of bioaccessible fumonisin B1 in different matrices afterin vitro digestion

Conventional analytical methods used for the analysis of fumonisin content in animal feeds fail to take into account the fumonisin content bound to the matrix, which is otherwise bioaccessible and can be absorbed from the gastrointestinal tract. Moreover, underestimation of fumonisin content using routine analytical methods can affect animal experiments using cereals contaminated by fungi. In the present study, hidden fumonisin B1 was analysed in two cereal substrates (maize and wheat) which were inoculated withFusarium verticillioides (MRC 826). The study compared a routine extraction procedure with anin vitro digestion sample pre-treatment. We found that all samples showed a higher content of fumonisin B1 after digestion, compared to the free fumonisin obtained only by extraction. The percentage of the hidden form was 38.6% (±18.5) in maize and 28.3% (±17.8) in wheat, expressed as the proportion of total fumonisin B1. These results indicate that the toxin exposure of the animals determined by the routine fumonisin analysis was underestimated, generally by 40%, as bioaccessibility was not taken into consideration. This is crucial in interpretation (and maybe in re-evaluation) of the results obtained from (other) animal experiments.

A true scale study of the maize chain with focus on free and hidden fumonisins and related fungi

The aim of this work was to study the dynamic profile of mycotoxin producing fungi as well as the accumulation of free and hidden fumonisins along the maize chain. Raw maize was sampled at harvest, at the storehouse and after drying. The study continued on the dried maize bulk along the storage period in a commercial silo, which was sampled during discharge at 6, 7, 8 and 10 months. Finally, the flour obtained after kernel milling and corresponding to the silo discharges was investigated. The sampling approach, designed in agreement with EU regulations and tested for reliability, was used along the maize chain, obtaining very good results. The prevalent fungi found in maize kernels belonged toFusarium sectionLiseola. Comparable results, in terms of both fungal incidence and fumonisin contamination, were obtained for the samples collected at harvest and at the storehouse, opening the possibility to adapt the maize chain monitoring to the needs of the farmer/enterprise. A reduction in fungal colony forming units was noticed after drying, supposedly due to the thermal treatment. Very similar amounts of free and hidden fumonisins were detected in raw maize and after drying, confirming the reliability of the sampling approach. During silo storage, up till to 10 months, a significant decrease was recorded for both free and hidden fumonisins. A significant increase in the free-to-total fumonisin ratio was observed moving from stored kernels to commercial flour, suggesting that milling leads to a reduction of the masked fraction. This result, together with the increase of free fumonisins after maize drying, should be considered in future studies. Especially the effect of drying conditions and milling treatments on the fumonisin masking phenomena should be further investigated.

Role of Maize Hybrids and Their Chemical Composition in Fusarium Infection and Fumonisin Production

This study was designed to investigate the role of hybrids in maize Fusarium section Liseola interaction and fumonisin production, with particular emphasis on the occurrence and accumulation of hidden fumonisins in maize (masking phenomenon). In this 2 year study, naturally infected field crops were chosen with 10 maize hybrids, six of them grown in both years. Maize samples collected in 2010 showed a higher incidence of fungal infection as well as higher fumonisin contamination than those obtained in 2009 but a very similar incidence of F. section Liseola. Fumonisin masking was confirmed in raw maize, with a lower amount of hidden forms as compared to free fumonisins detected in the year with higher contamination. The chemical composition of the different hybrids was determined and correlated with the contamination data: the results obtained highlight the main role of fatty acids, with a higher fumonisin contamination in hybrids showing a higher linoleic acid content and a higher masking action in hybrids with higher oleic to linoleic ratio. These results represent a good basis to explain maize hybrid susceptibility to F. section Liseola infection, fumonisin contamination, and masking not related to a specific commercial hybrid but extendable to all hybrids.

Free and bound fumonisins in gluten‐free food products

In this work a multiresidual LC-ESI-MS/MS method for the simultaneous detection of free and bound fumonisins is described, which allowed for a very low LOD and a very good recovery for all the analytes. The method was applied to the determination of free and bound fumonisins in several gluten-free products from the Italian market. Free fumonisins were found to occur in 90% of the samples: the overall median value was below the EU legal limit for foods for human consumption (800 microg/kg). Nonetheless, fumonisins occurred in several samples at concentrations above the legal limit, reaching also very strong contamination levels (maximum concentration level: 3310 microg/kg). Anyway, considering the limited diet of people suffering of the celiac disease or allergic to other wheat proteins, the incidence of fumonisin contamination may be envisaged as problematic. Furthermore, bound fumonisins were found to be present in all the analysed samples at similar or even higher amounts than the free forms. In many cases the sum of free and bound fumonisins exceeded the EU legal limit for total fumonisins also for those samples characterized by a low contamination of free fumonisins, thus opening a new important task to be addressed for the risk assessment in this field.

Production of a Single-Chain Variable Fragment Antibody against Fumonisin B1

The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free fumonisin B1 and fumonisin-biotin conjugate. Also fumonisin B2 was bound by the scFv. Modeling of both scFv and fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentially be used for developing a rapid and affordable immunoassay for detection of food contamination and can be applied in immunoaffinity chromatography, usually carried out prior to HPLC analysis of mycotoxin-contaminated food and feed.

Development of an improved monoclonal antibody‐based Elisa for fumonisin b1–3and the use of molecular modeling to explain observed detection limits

Monoclonal antibodies were prepared against the fumonisins, a group of mycotoxins produced by the plant pathogen, Fusarium moniliforme. Splenic lymphocytes, from Balb/c mice immunized with fumonisin B1‐ovalbumin conjugate, were fused with SP2/O myeloma cells, and 14 hybridomas were selected. In a competitive enzyme‐linked immunosorbent assay, fumonisin B1‐bovine serum albumin and free fumonisin B1 (FB1) competed for the monoclonal antibody. The concentrations of FB1 required to inhibit 50% antibody binding (IC50) ranged from 300 to 670 ppb. Antibodies also cross‐reacted with fumonisins B2 and B3 (FB2, FB3), and the hydrolyzed backbone of fumonisin B1 (HB‐FB1). None of the 14 monoclonal antibodies recognized the sphingolipids, sphingosine and sphinganine, that are structurally similar to the backbone of the fumonisins. Three‐dimensional computer models of FB1, FB2 and FB3 show the amine backbone folding with the two esterified trimethyl‐propane‐1,2,3‐tricarboxylic acid side‐chains to form a cage into which...

Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant.

Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant. Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used. In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective. Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant. A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding. The detection limit for fumonisin B1 in the ELISA was 100 ng/ml. The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid. Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively. These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.