Fumonisin B1, fumonisin B2, zearalenone and ochratoxin A contamination of maize in Croatia

Mycotoxins are products of moulds that frequently contaminate maize. In this study the presence of mycotoxins fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), zearalenone (ZEA) and ochratoxin A (OTA) was determined in 49 maize grain samples collected in autumn 2002. The most frequent finding was that of FB₁(100%), followed by ZEA (84%) and OTA (39%), while FB₂ was found only in three samples. The co-occurrence of two and three mycotoxins was found in 55 and 37% of samples, respectively. The concentrations (mean ± SD) of FB₁, ZEA and OTA in positive samples were 459.8 ± 310.7, 3.84 ± 6.68 and 1.47 ± 0.38 µg kg^-1, respectively, and the concentrations of FB₂ in three positive samples were 68.4, 109.2 and 3084.0 µg kg^-1. Although such low concentrations of mycotoxins are not a significant source of exposure in countries with a European diet, a few samples with extreme values indicate that thorough control is needed.     

A method for determining fumonisin B1 in corn using immunoaffinity column clean-up and thin layer chromatography/densitometry

A method for determining fumonisin B₁(FB₁) in corn was developed and the clean-up optimized in order to give an extract suitable for one-dimensional thin layer chromatographic (TLC) analysis. FB₁ was extracted with a solution of methanol:water (80:20,v/v), purified through an immunoaffinity column and separated on a C₁₈ reversed phase TLC plate. The FB₁ was visualized with 0.1mol/l sodium tetraborate, 0.40mg/ml fluorescamine in acetonitrile and 0.01mol/l boric acid:acetonitrile (2:3,v/v) for fluorescence detection, and quantified by densitometric analysis. Water, acetonitrile:water (1:1v/v) and acetonitrile:water (4:1v/v) were evaluated as TLC solvents for running both standards and samples together with derivatization procedures aimed at improving separation, resolution, sensitivity and linearity. The mean recovery for FB₁ for spiked samples was found to be 85% and the linear equation of standard calibration curve by densitometric analysis gave an r2 value higher than 0.99. The maximum coefficient of variation for replicate analysis of spiked samples was 19%. The absolute amount of FB₁ standard detectable on a TLC plate was 2 ng, giving a detection limit for the method of 0.1mg/kg. The method has been shown to be robust in the application of FB₁ monitoring in corn (214 samples) collected in different regions of the country. FB₁ was detected in 99% of these samples in the range of 0.2 to 6 mg/kg.     

Absorption, distribution and elimination of fumonisin B1 metabolites in weaned piglets

The absorption, distribution and elimination of fumonisin B₁ (and B₂) after oral administration of Fusarium verticillioides (MRC 826) fungal culture, mixed into the experimental feed for 10 days, was studied in weaned barrows. In order to determine the absorption of FB₁ from the feed marked by chromium oxide, a special T-cannula was implanted into the distal part of pigs' ileum. During the feeding of toxin-containing diet (45 mg FB₁ kg^-1) and until the tenth day after the end of treatment, the total quantity of urine and faeces was collected and their toxin content analysed. At the end of the trial, samples of lung, liver, kidney, brain, muscle, and fat were also collected and their fumonisin content analysed by LC-MS. The fumonisins appeared to decrease the reduced glutathione content in blood plasma and red blood cell haemolysate, possibly associated with in vivo lipid peroxidation. From a data set of 80 individual data and the concentration and rate of C_r and fumonisins (FB₁, partially hydrolysed FB₁ and aminopentol) in the chymus, it could be established that the accumulative absorption of fumonisin B₁ was 3.9% ± 0.7%. In the chymus, the FB₁ conversions into aminopentol and partially hydrolysed FB₁ were 1.0 and 3.9%, respectively. The degree of metabolism in faeces was variable, although the main product was the partially hydrolysed form, with very small amounts of the aminopentol moiety being recovered. In the investigated tissues the FB₁ conversion to aminopentol and partially hydrolysed FB₁ was 30 and 20%, respectively.     

In vitro microbial metabolism of fumonisin B1

There is a lack of information on the effect of swine caecal microbiota on fumonisin metabolism. In this in vitro study, the biotransformation of fumonisin B₁ (FB₁) by the gut microbiota of adult, healthy pigs was examined. Suspensions of caecal contents and McDougall buffer solution were incubated anaerobically with pure FB₁ for 0, 12, 24, 48 and 72 h. After 48 h, the conversion of FB₁ to partially hydrolysed FB₁ (46%) was nearly equal to the percentage ratio of FB₁, while by 72 h it was 49%. In vitro, the conversion of fumonisin B₁ to aminopentol was less than 1%. The results show that the caecal microbiota are capable of transforming fumonisin B₁ to the above metabolites. Further studies on FB₁ metabolism in the small intestine are clearly justified.     

Natural co-occurrence of aflatoxin B1, fumonisin B1 and ochratoxin A in barley and corn foods from Korea

A survey for aflatoxin B₁ (AFB₁), fumonisin B₁ and ochratoxin A (OTA) was conducted on 127 samples that included 30 food-grade barley, 32 barley foods, 18 food-grade corn and 47 corn foods, randomly collected during 1998-99 in Seoul, Korea. The presence of mycotoxins was analysed by direct competitive enzyme-linked immunosorbent assay (ELISA), and most of the positive samples from ELISA were confirmed using high-performance liquid chromatography (HPLC). Recoveries of AFB₁ and OTA spiked at 10 ng g -1 and FB₁ spiked at 50 ng g^-1 were 106, 87 and 105% by ELISA, whereas those by HPLC were 80, 79 and 84%, respectively. Detection limits by ELISA for AFB₁, FB₁ and OTA were 1, 5 and 5 ng g^-1, and those by HPLC were 0.6, 35 and 1 ng g^-1. Naturally occurring AFB₁, FB₁ and OTA were found in 4/32 (12%), 2/32(6%) and 4/32 (12%) samples of barley foods with an average of 26, 16 and 9 ng g^-1, respectively. AFB₁ and FB₁ in corn foods were detected in 4/47 (8%) and 9/47 (19%) samples with the average being 20 and 74 ng g^-1, while no OTA was found in any corn foods samples. No AFB₁, FB₁ or OTA was detected in any of food-grade barley and corn samples. This is the first report on the natural co-occurrence of AFB₁ and FB₁ in barley and corn foods as well as on surveillance of OTA in Korea.     

Survey for fumonisin B1 in Korean corn-based food products

Seventy-six corn-based foods collected in Seoul, Korea, including corn flakes, corn snack, cornstarch, corn for popping, roasted corn for tea, canned sweet corn and other corn products were analysed for the occurrence of fumonisin B₁ (FB₁) by using direct competitive enzyme-linked immunosorbent assay (dcELISA) and high-performance liquid chromatography (HPLC). The average recoveries of FB₁ from the corn flakes sample in the range 5-1000 ng g^-1 were 104% by dcELISA and 82% by HPLC. The limits of detection were approximately 5 ng g^-1 by dcELISA and 20 ng g^-1 by HPLC. The incidences and mean levels of FB₁ were 73.3, 78.6, 50, 58.3, 17.6 and 40% and 41.8, 67.9, 114, 256, 172 and 22 ng g^-1 from corn flakes, corn snack, corn starch, corn for popping, roasted corn for tea and other corn products, respectively, by dcELISA. No FB₁ was found in canned sweet corn. The results obtained by dcELISA were correlated to those by HPLC for FB₁ (r² = 0.992). This is the first report on the occurrence of FB₁ in corn-based foods in Korea.     

Occurrence of fumonisins B1 and B2 in Portuguese maize and maize-based foods intended for human consumption

Fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) are mycotoxins mainly produced by Fusarium verticillioides and Fusarium proliferatum, which are field pathogens of maize. A survey was conducted on the incidences of FB₁ and FB₂ in both maize and derived products purchased in Portugal. The analytical method involved extraction with methanol-water, clean-up by immunoaffinity column and derivatization with naphthalene-2,3-dicarboxaldehyde. Determination was carried out by high-performance liquid chromatography (HPLC) with spectrofluorimetric detection, with liquid chromatography/mass spectrometry (LC/MS) confirmation. The presence of FB₁ and FB₂ was determined in 67 samples of maize and maize-based foods, such as flour, semolina, starch, sweet maize, cornflakes and other breakfast cereals, and snacks collected in 2005. FBs were found in 15 samples at concentrations ranging from 113 to 2026 µg kg^-1. Two of the samples showed higher contamination levels than the limits established by the European Commission Regulation. None of the samples contained levels of fumonisins that would lead to an exposure exceeding the tolerable daily intake (TDI).     

Fumonisin levels in commercial corn products in Buenos Aires, Argentina

Fumonisins B₁ (FB₁) and B₂ (FB₂) were determined in 35 samples of corn flour and corn grits destined for human consumption and purchased directly from Buenos Aires food shops and supermarkets from October 1996 to January 1997 and during the month of January 1998. During the first period of sample collecting, 16 out of 19 samples were found to be contaminated. Considering all 19 samples, contamination levels were between not detected and 1860 ng/g FB₁, and from not detected to 768 ng/g FB₂. During the second period all 16 samples were found to be contaminated with levels ranging from 75 to 4987 ng/g FB₁, and from not detected to 1818 ng/g FB₂. The levels of FB₁ and FB₂ in the samples collected during January 1998 were significantly higher than the samples collected during the period from October 1996 to January 1997. No significant difference was found in terms of fumonisin levels between the branded and unbranded samples.     

Determination of fumonisins B1 and B2 in cornflakes by high performance liquid chromatography and immunoaffinity clean-up

The determination of fumonisins in cornflakes is a challenging matter as the actually available methods for the analysis of corn do not perform well when applied to this more complex matrix. After testing several factors that may affect the analytical performance, an accurate method for the determination of fumonisin B₁ (FB₁) and B₂ (FB₂) in cornflakes has been developed. The method uses immunoaffinity chromatography for clean-up and high performance liquid chromatography (HPLC) for quantification of the toxins. Samples were extracted twice with acetonitrile-methanol-water (25:25:50) and the combined extracts were diluted with phosphate buffered saline (PBS) and applied to a FumoniTest? immunoaffinity column. After washing with PBS, fumonisins were eluted from the column with methanol and reacted with o-phthaldialdehyde/2-mercaptoethanol to form fluorescent derivatives. Fumonisin derivatives were analysed by reversed phase HPLC with fluorometric detection using methanol-0.1 M phosphate buffer (77:23; pH adjusted at 3.35) as mobile phase. The average recoveries for FB₁ and FB₂     

Fumonisins B1 and B2 in Brazilian corn-based food products

Eighty-one samples of corn products were acquired from markets and supermarkets in the city of Campinas, SP, Brazil, and were analysed for fumonsins B₁ and B₂ (FB₁ and FB₂). Forty samples (49%) were positive for FB₁     

Natural co-occurrence of deoxynivalenol and fumonisins B1 and B2 in Italian marketed foodstuffs

A survey was performed to obtain the frequency and levels of contamination by deoxynivalenol (DON) and fumonisins B₁ and B₂ (FB1, FB2) mycotoxins in Italian marketed foods. Of 202 samples investigated, including raw materials and processed cereal foods (bread, pasta, breakfast cereals, biscuits, baby and infant foods), 84% were contaminated with DON at levels from 0.007 to 0.930 μg g^-1 (median 0.065 μg g^-1); 26% contained FB1 ranging from 0.010 to 2.870 μg g^-1 (0.070 μg g^-1); 35% contained FB2 at 0.010-0.790 μg g^-1 (0.080 μg g^-1). The highest levels of DON and FB1 were detected in raw cereals and wholemeal flours. The highest levels of FB2 were detected in durum wheat pasta. A widespread DON contamination was found in baby and infant foods at levels varying from 0.007 to 0.166 μg g^-1.     

Linking extraction and purification of maize samples for fumonisin analysis

Fumonisins are produced by several fungal species that are common contaminants of maize, The most abundant naturally occurring fumonisin, fumonisin B₁ (FB₁), has been shown to induce several animal disease syndromes. The development of analytical methods is therefore important. A new method is described that integrates extraction and purification of maize samples in one step. It efficiency is compared against wellknown methods, and shows similar results for naturally contaminated maize. It is concluded that the proposed method can be applied to fumonisin B₁ and fumonisin B₂ (FB₂) analysis in maize at least within the con2 centration range found.     

Analysis of heat-processed corn foods for fumonisins and bound fumonisins

Thirty retail samples of heat-processed corn foods, i.e. corn flakes, corn-based breakfast cereals, tortilla chips and corn chips, were analysed for fumonisins — fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and hydrolysed FB₁ (HFB₁) — as well as for protein- and total-bound FB₁. Bound (hidden) fumonisins cannot be detected by conventional analysis. Improved methods for the determination of bound FB₁ were developed. The protein-bound FB₁ was extracted with 1% sodium dodecylsulfate (SDS) solution. The SDS, which interfered with high-performance liquid chromatography (HPLC) analysis, was then separated from protein-bound FB₁ by complexing with methylene blue followed by solvent extraction and hydrolysis with 2 N KOH. To measure total-bound FB₁, the sample itself was hydrolysed with KOH. In both cases, clean-up was accomplished on an OASIS polymeric solid-phase extraction column and the bound fumonisins were determined by HPLC measurement of HFB₁. Fourteen of 15 samples of corn flakes and other corn-based breakfast cereals analysed contained detectable levels of FB₁ with a mean in positive samples of 67 ng g^-1 (13-237 ng g^-1). Two samples also had detectable levels of FB₂ (21-23 ng g^-1). Bound FB₁ was found in all samples; the mean protein-bound FB₁ measured was 58 ng g^-1 (22-176 ng g^-1) and the mean total-bound FB₁ measured was 106 ng g^-1 (28-418 ng g^-1), reported as FB₁ equivalents after correction for recoveries of HFB₁. There was an average of about 1.3 times more FB₁ in the bound form compared with extractable FB₁, and this was about twice as much as protein-bound FB_1. Seven of the 15 samples of alkali-processed corn-based foods, such as tortilla chips and corn chips, contained FB₁ and three contained HFB₁ with means in measurable positive samples of 78 (48-134) and 29 (13-47) ng g^-1, respectively. Five of these alkali-processed corn foods contained bound FB₁; the mean measurable protein-bound FB₁ was 42 ng g^-1 (39-46 ng g^-1) and the mean measurable total-bound FB₁ was 100 ng g^-1(54-209 ng g^-1). HFB₁ derived from bound FB₁ in selected samples was confirmed by HPLC with mass spectrometry (MS).     

Determination of the mycotoxin fumonisin B1 in maize by reversed-phase thin-layer chromatography: a collaborative study

A simple and cost-effective method using thin-layer chromatography for the determination of the mycotoxin fumonisin B₁ in maize is described. The analytical method consisted of the extraction of ground maize by shaking with methanol/water (75:25) for 60 min and clean-up of the resultant extract by means of strong anion exchange solid-phase extraction. The purified residue, formed by evaporation of the elution solvent, was reacted with fluorescamine and the fumonisin B₁-derivative was separated by reversed-phase thin-layer chromatography using a developing solution of methanol/aqueous 4% potassium chloride (70:30). The derivatized FB₁ was readily visualized as a greenish-yellow spot under long wavelength ultraviolet light and quantified by visual comparison with a set of similarly derivatized standards in the range 20-300 ng FB₁ spotted on plate. Based on visual comparison, levels down to 0.5 mg kg^-1 were successfully estimated. The method was collaboratively studied in 14 laboratories using four duplicate maize meal samples (including a blank) and a spiked sample for determination of recovery. No significant difference was observed between mean FB₁ levels by high-performance liquid chromatography or thin-layer chromatography. Based on within-laboratory relative standard deviations of 27.1-41.7% and between-laboratory relative standard deviations of 35.0-63.3%, the method can be considered semiquantitative. The mean recovery achieved by participants at a spiking level of 2.00 mg kg^-1 was 74.5%.     

Determination of sphinganine, sphingosine and Sa/So ratio in urine of humans exposed to dietary fumonisin B1

Fumonisin B₁ (FB₁) is an inhibitor of sphinganine N-acyltransferase and the increase in the sphinganine/sphingosine (Sa/So) ratio in urine or serum has been proposed as a biomarker to evaluate exposure to fumonisins. The objectives of this study were to (1) develop a liquid chromatographic method sufficiently sensitive to determine the low concentration of free Sa in male human urine, and (2) analyse So and Sa in human urine and monitor the Sa/So ratio in urine of humans exposed to FB₁ in corn diets over 1 month. The liquid chromatographic method involved isolation from human urine of exfoliated cells followed by an extraction of free sphingoid bases and their separation and quantification by high performance liquid chromatography. The detection limits for So and Sa were 0.15ng/ml in female urine (2ml used) and 0.005ng/ml in male urine (60ml used). Twenty-eight healthy adult volunteers consumed for 1 month a normal diet containing their homegrown corn potentially contaminated with FB₁. Immediately preceding the start of the test, morning urine samples for the determination of So and Sa were collected from each person, and the corn samples used in cooking were obtained from each family for the determination of FB₁. At the end of the test period, morning-urine samples were collected from each person and analysed again. The daily FB₁ intakes were estimated and used to assess the relationship between them and the urinary Sa/So ratios in humans exposed to dietary FB₁ over 1 month. All the home grown corn samples contained FB₁ ranging from 0.08 to 41.1mg/kg, and the estimated daily FB₁     

Occurrence of fumonisins B1 and B2 in asparagus from Shandong province, P.R. China

Thirty samples of asparagus spears were collected from the fields in Shandong province, China, in July 2004, and were analysed for the occurrence of fumonisins B₁ and B₂ (FB₁ and FB₂) by HPLC coupled with electrospray ionization tandem mass spectrometry. Twenty-four samples (80%) contained fumonisins, ranging from 24 to 670 ng g^-1 (average 123 ng g^-1) and 17 to 138 ng g^-1(average 35 ng g^-1) for FB₁ and FB₂, respectively. The total amount of fumonisins (FB₁ and FB₂) in all samples ranged from 47 to 714 ng g^-1 (average 158 ng g^-1) (based on dry weight). This is the first report on the natural occurrence of FB₁ and FB₂ in asparagus spears in China.     

Comparison of different extraction and clean-up procedures for the determination of fumonisins in maize and maizebased food products

In order to optimize the analytical method for the determination of fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in diVerent maize products, five materials (maize flour, cornflakes, extruded maize, muffins and infant formula) were investigated under a variety of experimental conditions organized in a ruggedness test according to a factorial design. The influence of five factors (extraction solvent, extraction mode, volume of extraction solvent, test sample size and clean-up) on method performances was tested by four laboratories using spiked materials (0.5 μg/g and 1.5 μg/g FB₁ + FB₂) and naturally contaminated materials (ca 1.5 μg/g with FB₁ + FB₂). The end determination step was performed by high-performance liquid chromatography analysis of the o-phthaldialdehyde derivatized extracts. The ruggedness test permitted identification of two critical factors in the analysis of fumonisins in the above products, namely 'extraction solvent' and 'cleanup procedure'. In particular, the use of acetonitrile (ACN)-water (1 + 1, v + v) as extraction solvent and immunoaffinity column for clean-up provided better recovery of fumonisins and chromatographic resolution as compared with methanol (MeOH)-water (3 + 1, v + v) and strong anion exchange (SAX), respectively. However, phase separation occurring after extraction with ACN-water may have given incorrect results. Based on the information obtained with the present study it was possible to develop a new method horizonhorizontally applicable to all the above mentioned maize-based food matrices.     

Naturally occurring phenols: a detoxification strategy for fumonisin B1

Phenolic compounds from plants offer a means for both the prevention and detoxification of mycotoxins that affect human health. This research investigates the control of fungal growth and toxin production by Fusarium verticillioides with plant phenolic compounds, namely chlorophorin, iroko and maakianin, benzoic acid, caffeic acid, ferulic acid, and vanillic acid. Inhibition by these compounds of fungal growth was determined by the agar overlay method and their effect on fumonisin B₁ (FB₁) production was determined by high-performance liquid chromatography. Chlorophorin was the most effective compound in inhibiting fungal growth, followed by iroko, maakianin, vanillic acid and caffeic acid. Chlorophorin also was the most effective compound in reducing toxin production (94% reduction), followed by caffeic acid, ferulic acid, vanillic acid and iroko, which reduced FB₁ levels by 90-91%. The widespread occurrence of fumonisins world-wide and the lack of adequate prevention of fumonisins require 'biologically safe' alternatives to prevent the transfer of fungi and their health hazardous toxins into our daily foods and environment.     

Tissue sphinganine as a biomarker of fumonisin-induced apoptosis

NCTR measured sphinganine concentrations in the livers of mice and in the livers and kidneys of rats in conjunction with a tumour bioassay. In our model of the tumour incidence, target-tissue levels of sphinganine serve as a biomarker for a dose response of fumonisin B₁ on cell death. Initially we questioned the utility of sphinganine levels in this role because they were highly variable when compared across time points. In spite of this concern, a conceptual framework and data are presented that support the use of sphinganine as a biomarker for a dose response of fumonisin B₁ on cell death. This framework is reasonably consistent with observed sphinganine concentrations in the examined tissues, the literature on fumonisin's effects on sphingolipid synthesis, and our hypothesized mechanism through which fumonisin B₁ increases age-specific tumour incidence.     

Residue formation of fumonisin B1 in porcine tissues

The residues derived from the uptake of fumonisin B1, a toxic metabolite of Fusarium verticillioides frequently occurring in corn and corn products, were determined in growing pigs. After oral administration of 100 mg FB₁/animal/day for 5-11 days, serum, bile, lung, liver, kidney, brain, spleen, pancreas, heart, muscle, eye, and fat samples were collected immediately and analysed by LC-MS. The highest values were measured in kidney (833 ±1329 μg kg^-1, mean ±SD), liver (231 ±163 μg kg^-1), lung (170 ±311 μg kg^-1) and spleen (854 ±2212 μg kg^-1). Muscle contained 26 ±41 μg kg^-1, while in fat only 2 ±3 μg kg^-1 were traceable. Despite the potential accumulation over extended feeding periods as well as the large variations in the residue formation of FB₁, a carry-over in edible tissues from swine was considered not to be of toxicological relevance.