HPLC determination of fumonisin mycotoxins in maize: a comparative study of naphthalene-2,3-dicarboxaldehyde and o-phthaldialdehyde derivatization reagents for fluorescence and diode array detection

Fumonisins are mycotoxins produced by various species of Fusarium and occur naturally in contaminated maize and maize-based foods. Ingestion of fumonisins has considerable health implications for humans and animals. Since fumonisins lack a useful chromophore or fluorophore, their determination in maize is routinely achieved via HPLC with fluorescence detection (FLD) after precolumn derivatization. This study optimized naphthalene-2,3-dicarboxaldehyde (NDA) derivatization of fumonisins in naturally contaminated maize following strong anion exchange (SAX) solid phase extraction (SPE) clean-up and utilizing diode array detection (DAD) as a practical alternative simultaneously to FLD. The limit of detection (LOD) for fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) with FLD was 0.11 ng, 0.50 ng and 0.27 ng, respectively, and with DAD it was 13.8 ng, 12.5 ng and 6.6 ng, respectively injected on column. The coefficient of variation (CV, n = 6) for FB(1), FB(2) and FB(3) in a naturally contaminated samples obtained with FLD was 2.6%, 1.8% and 5.3%, respectively, compared to 6.0%, 3.4% and 9.5%, respectively, obtained with DAD. Subsequently the optimized NDA derivatization was compared to the widely used o-phthaldialdehyde (OPA) derivatization agent as well as alternative sample clean-up with immunoaffinity column (IAC) by analyzing naturally contaminated maize samples (n = 15) ranging in total fumonisin (TFB = FB(1)+FB(2)+FB(3)) levels from 106 to 6000 μg/kg. After immunoaffinity column clean-up of extracted samples, the recoveries of spiked maize samples for NDA-FLD of FB(1), FB(2) and FB(3) were 62%, 94% and 64%, respectively. NDA proved to be an effective derivatization reagent of fumonisin in naturally contaminated maize samples following IAC clean-up, except for DAD at TFB levels below 1000 μg/kg. In contrast NDA derivatization following SAX clean-up produced results comparable to OPA only for levels below 1000 μg/kg. Aside from the difference in detection limits, FLD and DAD produced comparable results irrespective of the clean-up method or the derivatization agent.     

Fiber-optic immunosensor for mycotoxins

Evanescent wave-based fiber-optic immunosensors were studied for the detection of fumonisins and aflatoxins in maize. Two formats, competitive and non-competitive, were used. A competitive format was used to measure fumonisin B1 (FB1) in both spiked and naturally contaminated maize samples. Fumonisin monoclonal antibodies were covalently coupled to an optical fiber and the competition between FB1 and FB1 labeled with fluorescein (FB1-FITC) for the limited number of binding sites on the fiber was assessed. The signal generated in the assay was inversely proportional to the FB1 concentration. For samples, the concentration causing an inhibition of binding by 50% (IC50) was dependent upon the clean-up procedure used. Simple dilution of methanolic maize extracts yielded an assay with an IC50 equivalent to 25 microg FB1 g(-1) maize with a limit of detection of 3.2 microg g(-1) maize. Affinity column clean-up yielded an assay with an IC50 equivalent to 5 microg FB1 g(-1) maize (limit of detection 0.4 microg FB1 g(-1)). An HPLC method and the immunosensor method agreed well for naturally contaminated maize samples except when large amounts of other fumonisins that cross-react with the immunosensor were present. The second sensor format, for the mycotoxin aflatoxin B1 (AFB1), was a non-competitive assay using the native fluorescence of this mycotoxin. Because the fluorescence of AFB1 itself was detected, the response of the sensor was directly proportional to the toxin concentration. The sensor, while capable of detecting as little as 2 ng ml(-1) of AFB1 in solution was technically not an immunosensor, since the attachment of aflatoxin specific antibodies was not required. Sensors of the formats described have the potential to rapidly screen individual maize samples but require coupling with a clean-up technique to be truly effective.     

Identification and heritability of fumonisin insensitivity in Zea mays

Landraces of maize (Zea mays ssp. mays) and its wild teosinte relatives (Zea mays spp. parviglumis and mexicana) were surveyed for sensitivity to fumonisin B(1), a phytotoxin produced by the maize pathogen Gibberella moniliformis. Only two of 42 Z. mays samples were highly insensitive to FB(1) (ED(50) = ca. 200 microM). The teosintes and 76% of the maize landraces were moderately or highly sensitive to FB(1) (ED(50) < or = 30 microM), which indicates that FB(1) sensitivity is likely to be an ancestral trait in Z. mays. F(1) generations derived from crosses between FB(1)-sensitive maize inbred B73 and insensitive landraces were significantly less sensitive than B73. Thus, our data indicate that FB(1)-insensitivity is a relatively rare but heritable trait in maize. We also report the sensitivity of maize to other Gibberella toxins - beauvericin, diacetoxyscirpenol, and moniliformin.     

Analysis of Fusarium toxins in maize and wheat using thin layer chromatography

Thin layer chromatography (TLC) methods for identifying and quantifying deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone in grain samples were compared to immunoassay (ELISA) and high performance liquid chromatography (HPLC) methods to determine the reliability of the less expensive TLC. There was a very good agreement between levels of DON measured by TLC and competitive-direct ELISA, and between levels of fumonisin B1 measured by TLC and HPLC, over a wide range of concentrations. Correlation coefficients (Pearson's) were 0.978, 0.914 and 0.953 for DON in maize, DON in wheat and FB1 in maize respectively. A lower correlation coefficient (r = 0.672) was obtained when zearalenone was quantified by TLC and HPLC. Possible reasons for this are discussed. A cost comparison of the various methods revealed that TLC was the least expensive for sample analysis. It is recommended that researchers choose which analytical method to use based upon individual considerations of cost and precision. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fumonisins production by Fusarium proliferatum strains isolated from asparagus crown

The present work deals with the capability for producing fumonisin by Fusarium proliferatum strains isolated from asparagus in China. Fifty of F. proliferatum strains were randomly selected and incubated on cultures of maize grain and asparagus spear, respectively. Fumonisin levels (FB₁ and FB₂) were determined by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that all 50 strains produced fumonisins in maize culture within a wide range of concentrations, 10–11,499 μg/g and 2–6,598 μg/g for FB₁ and FB₂, respectively. On culture of asparagus spear,48 strains (96%) produced fumonisins in the range 0.2–781.6 μg/g and no detected to 40.3 μg/g for FB₁ and FB₂, respectively. All of F. proliferatum strains produced much higher levels of FB₁, FB₂ and total fumonisins (FB₁ + FB₂) in maize grain culture than in asparagus spear culture. Meanwhile, fumonisin B₃ (FB₃) was identified in all maize culture extracts and most of asparagus spear culture extracts. This is the first study carried out the fumonisin-producing ability of F. proliferatum strains isolated from asparagus in China. The information obtained is useful for assessing the risk of fumonisins contamination in asparagus spear. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Fumonisin occurrence in corn from high- and low-risk areas for human esophageal cancer in China.

Forty-seven corn samples were collected in 1989 from Linxian and Shangqiu Counties in Henan Province, the high- and low-risk areas, respectively, for human esophageal cancer in the People's Republic of China. The samples were analyzed for fumonisin (fumonisin B1 [FB1] and FB2) contamination. Of the fumonisin-positive samples, the mean levels in Linxian corn were found to be 872 ng/g for FB1 and 448 ng/g for FB2, while the Shangqiu corns had 890 ng of FB1 and 330 ng of FB2 per g. The incidence of fumonisin contamination of Linxian corn (48%) was about two times higher than that of Shangqiu corn (25%), and the former corn samples were frequently cocontaminated with trichothecenes. Fusarium species isolated from corn from Linxian County produced FB1 at levels ranging from 1,280 to 11,300 micrograms/g.     

Fumonisin mycotoxins in human hair

This study shows for the first time the accumulation of fumonisin mycotoxins in human hair of population clusters exposed to contaminated maize, and thus the feasibility of human hair analysis for the assessment of past fumonisin exposure. Composite hair samples were obtained from the Bizana, Butterworth and Centane districts within the Transkei region of the Eastern Cape Province of South Africa. Following methanol extraction and strong anion exchange clean up, the fumonisins FB₁, FB₂ and FB₃ were detected using high performance liquid chromatography coupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS). Hair from Centane and Butterworth showed mean levels of FB₁ of 26.7 and 23.5 μg kg^-1 hair, respectively. FB₂ was only detected in hair from Centane and in one sampling point in Butterworth, with mean levels of 6.5 and 5.7 μg kg^-1 hair, respectively. Hair samples from Bizana, on the other hand, were found to contain higher levels of FB 1 (mean 33.0 μg kg^-1 hair) and FB 2 (mean 11.1 μg kg^-1 hair). No samples contained more than trace levels of FB 3 . Recoveries from spiked hair samples using this method ranged from 81% to 101%, demonstrating the applicability of hair analysis in assessing human exposure to fumonisin mycotoxins.     

Detection and quantification of fumonisins from <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> in maize grown in southern India

Fumonisins are a group of fungal toxins, occurring worldwide in maize infected mainly by Fusarium verticillioides. This paper describes the level of fumonisins in maize seed samples and the ability of F. verticillioides strains isolated from maize seeds grown in India to produce fumonisins. Forty-three seed samples intended to be used for consumption were collected from different regions of Karnataka and Andhra Pradesh. The samples were subjected to the agar plate method for the detection of F. verticillioides. Identification of F. verticillioides was done based on morphological characters and further confirmed by polymerase chain reaction. The majority of the samples were infected by F. verticillioides and infection percentage in the individual samples ranged from 5 to 51%. Twenty-three out of 35 (65%) strains were positive for fumonisin production in high performance liquid chromatography (HPLC) and competitive direct-enzyme linked immuno sorbent assay (CD-ELISA). Fumonisin level in seed samples ranged from 200 to 1,722 μg/g using CD-ELISA. HPLC could differentiate FB1 and FB2 toxins; out of 35 strains, 14 (40%) showed both FB1 and FB2 production. These findings indicate that there may be a risk of human exposure to fumonisins through the consumption of F. verticillioides infected corn-based foods in India.     

Fumonisin mycotoxins in human hair

This study shows for the first time the accumulation of fumonisin mycotoxins in human hair of population clusters exposed to contaminated maize, and thus the feasibility of human hair analysis for the assessment of past fumonisin exposure. Composite hair samples were obtained from the Bizana, Butterworth and Centane districts within the Transkei region of the Eastern Cape Province of South Africa. Following methanol extraction and strong anion exchange clean up, the fumonisins FB₁, FB₂ and FB₃ were detected using high performance liquid chromatography coupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS). Hair from Centane and Butterworth showed mean levels of FB₁ of 26.7 and 23.5 μg kg^?1 hair, respectively. FB₂ was only detected in hair from Centane and in one sampling point in Butterworth, with mean levels of 6.5 and 5.7 μg kg^?1 hair, respectively. Hair samples from Bizana, on the other hand, were found to contain higher levels of FB 1 (mean 33.0 μg kg^?1 hair) and FB 2 (mean 11.1 μg kg^?1 hair). No samples contained more than trace levels of FB 3 . Recoveries from spiked hair samples using this method ranged from 81% to 101%, demonstrating the applicability of hair analysis in assessing human exposure to fumonisin mycotoxins.     

Ochratoxin A and fumonisins (B1 and B2) in maize from Balkan nephropathy endemic and non endemic areas of Croatia

The occurrence of ochratoxin A, fumonisin B1 and B2 has been investigated in maize samples collected in 1996 (105 samples) and 1997 (104 samples) in 14 counties of Croatia, including Brodsko-Posavska county, the main area of Balkan endemic nephropathy in Croatia. Ochratoxin A and fumonisins co-occurred in 21% of the examined samples. In particular, ochratoxin A (OTA) was found in 10 samples (10%) of the 1996 and 36 samples (35%) of the 1997 crops with mean concentrations of positive samples of 37.9 ng/g and 57.1 ng/g, and highest concentrations at 223.6 ng/g and 613.7 ng/g, respectively. Similar incidence of OTA contamination was observed in 1996 samples from both endemic and non endemic areas of Balkan nephropathy, whereas a significant difference (P<0.01) was found between the two areas in 1997, with 50% and 20% incidence of contamination in the endemic and non endemic area, respectively, and relevant OTA mean concentration of positive samples of 73.4 ng/g and 20.2 ng/g. High incidence of infection byPenicillium spp. (potential OTA producers) was found in all tested samples, with mean values of 88% and 93% in samples of 1996 and 1997, respectively. With respect to fumonisin B1 (FB1) and B2 (FB2) all but one of the 1996 samples were contaminated, with highest and mean concentrations of positive samples (FB1+FB2) at 11661 ng/g and 645 ng/g, respectively. Similar incidence of positive samples (93%), but lower contamination levels (mean 134 ng/g, maximum 2524 ng/g) were found in 1997 samples. The results of fumonisin analysis were in agreement with the mycological analysis showing higher incidence of Fusarium infection in samples of 1996 with respect to those of 1997. These data provide additional information on the occurrence of ochratoxin A in Balkan endemic nephropathy areas and, for the first time, its co-occurrence with other nephrotoxic compounds, such as fumonisins, that may contribute to the disease development. However the finding of these mycotoxins in the non-endemic areas, also at high levels, do not allow to draw a conclusion about their role in the etiology of the disease.     

Simple method for the simultaneous isolation and determination of fumonisin B1 and its metabolite aminopentol-1 in swine liver by liquid chromatography--fluorescence detection

An analytical method based on high-performance liquid chromatography (HPLC) combined with fluorescence detection (FL) has been developed for the simultaneous determination of fumonisin B1 (FB1) and its totally hydrolized metabolite aminopentol-1 (AP1) in pig liver. The sample preparation is based on a single solid phase extraction (SPE). o-Phthalaldehyde (OPA) was used for pre-column derivatization before the programmed reversed-phase analysis on phenylhexyl column. The developed method shows good repeatibility for inter- and intra-day precision as well as adequate linearity of calibration curves (r2 was 0.9855 for FB1 and 0.9831 for AP1). Average recoveries from the matrix were 93.6% for FB1 and 95.3% for AP1. The limit of quantification (LOQ) in swine liver was 75 microg/kg for FB1 and 42 microg/kg for AP1.     

Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels

Fusarium verticillioides, a fungal pathogen of maize, produces fumonisin mycotoxins that adversely affect human and animal health. Basic questions remain unanswered regarding the interactions between the host plant and the fungus that lead to the accumulation of fumonisins in maize kernels. In this study, we evaluated the role of kernel endosperm composition in regulating fumonisin B1 (FB1) biosynthesis. We found that kernels lacking starch due to physiological immaturity did not accumulate FB1. Quantitative polymerase chain reaction analysis indicated that kernel development also affected the expression of fungal genes involved in FB1 biosynthesis, starch metabolism, and nitrogen regulation. A mutant strain of F. verticillioides with a disrupted a-amylase gene was impaired in its ability to produce FB1 on starchy kernels, and both the wild-type and mutant strains produced significantly less FB1 on a high-amylose kernel mutant of maize. When grown on a defined medium with amylose as the sole carbon source, the wild-type strain produced only trace amounts of FB1, but it produced large amounts of FB1 when grown on amylopectin or dextrin, a product of amylopectin hydrolysis. We conclude that amylopectin induces FB1 production in F. verticillioides. This study provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.     

A survey on the occurrence of mycotoxins in wheat and maize from western Romania

Samples of wheat (n = 25) and maize (n = 30) for animal consumption, collected in 1997 after harvest from western Romania, were analyzed by enzyme immunoassays for mycotoxin contamination. Toxins analyses included deoxynivalenol (DON), 3-acetylDON, 15- acetylDON, fusarenone X (FX), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), zearalenone (ZEA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), ochratoxin A (OA), and citrinin (CT). DON and acetylDONs were the major contaminants in wheat (100%) and maize (46%). Median values for DON, 3-acetylDON, and 15-acetylDON were 880 μg kg-1, 66 μg kg- 1, and 150 μg kg-1 in wheat, and 890 μg kg-1, 180 μg kg-1, and 620 μg kg- 1 in maize, respectively. Additionally, 3,15-diacetylDON was detected in some samples by HPLC-EIA analysis. All samples were negative for FX (<150 μg kg-1). T-2 was found in wheat (n = 6) and maize (n = 1) at levels between 13 and 63 μg kg- 1. DAS (2.6 μg kg-1) was found in one maize sample. ZEA occurred in all wheat and in four maize samples, median values were 10 μg kg-1 and 250 μg kg-1, respectively. One maize sample contained FB1 (140 μg kg-1). All samples were AFB1-negative (<4 μg kg-1). OA was found in one wheat sample (37 μg kg- 1), CT was found in one maize sample (580 μg kg- 1). This first reported natural occurrence of a range of mycotoxins in Romanian feeding stuff shows that DON and acetyl DONs may be present at levels which may affect animal production. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrogen repression of fumonisin B1 biosynthesis in Gibberella fujikuroi

Fumonisins are a group of structurally related mycotoxins produced by Gibberella fujikuroi. The fungus produced fumonisin B1 (FB1) as early as 18 hour in a defined medium containing 1.25 mM or 2.5 mM ammonium phosphate, whereas fumonisin B1 production was repressed for 75 hour and 125 hour when mycelia were resuspended in media containing ammonium phosphate at 10 mM or 20 mM, respectively. Although total fumonisin B1 production was greater in resuspension cultures grown in higher concentrations of ammonium phosphate, the accumulation was independent of the inoculum size and carbon/nitrogen ratio. The addition of ammonium phosphate to cracked corn cultures also repressed fumonisin B1 production by 97%, and persisted for at least three weeks. Thus, biosynthesis of fumonisin B1 is regulated by a mechanism involving nitrogen metabolite repression, suggesting that control strategies that target the regulatory elements of nitrogen metabolism may be effective at reducing the risk of fumonisin contamination in food.     

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.     

Selective sample cleanup by immunoaffinity chromatography for determination of fenvalerate in vegetables

This paper describes the establishment of an immunoaffinity chromatography (IAC) for selective extraction of fenvalerate from vegetable samples. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against fenvalerate to CNBr-activated Sepharose 4B and packed into a cartridge. The extraction conditions were carefully optimized, including loading, washing and eluting solutions. Under the optimal conditions, the IAC column was able to capture fenvalerate with the maximum capacity of 4000 ng. An average recovery of 94.5% and a RSD of 8.8% were obtained with six IAC columns prepared on six different days. Three vegetable samples spiked with fenvalerate at four different concentrations were extracted with IAC column and determined by gas chromatography with electron capture detection (GC-ECD). Chromatograms of final extracts were clean and fenvalerate could be easily detected without the interferences. The extraction recoveries and RSD were 74.7-96.5% and 2.5-5.2%, respectively, and the calculated limit of detection of the whole method was 0.008-0.012 ng g(-1).     

Accumulation of fumonisins B1 and B2 in freshly harvested Brazilian commercial maize at three locations during two nonconsecutive seasons

Fifty-six Brazilian commercial maize cultivars were examined for FB1 and FB2 accumulation after two non-consecutive growing seasons. During the 94/95 growing season 35 cultivars were planted at three locations in the state of S?o Paulo, Brazil. All samples (total of 105) were contaminated (0.10 micro/g-6.58 microg/g FB1 and 0.04 microg/g-2.15 microg/g FB2). During the 97/98 growing season, 8 of the cultivars used during 94/95 and 21 others were replanted at the same locations. All 87 samples were contaminated (1.15 microg/g-43.80 microg/g FB1 and 0.08 microg/g-11.65 microg/g FB2). One cultivar accumulated significantly less fumonisins in all locations during both growing seasons, indicating that some degree of selection may be possible even in climates that favor F. moniliforme (verticillioides) infection of maize. The presence of water surplus in soil from kernel maturity to harvest correlated with concentrations of FB1 in the grain for the 8 cultivars planted during both seasons at three locations. Observed trends indicated that water excesses and deficits from silking to harvest increased fumonisin levels. The difference in the incidence of FB1, FB2, and FB1 + FB2 was significant between growing seasons, planting locations and between cultivars. Neither the level of hybridization, nor the type of endosperm, nor the length of the vegetative cycle showed any effect on the FB1 contamination.     

Effect of extraction and extract purification on the measurable fumonisin content of maize and maize products. Tests on the efficiency of acid extraction and use of immunoaffinity columns

The extraction of naturally contaminated maize-based products using methanol / water (3+1) or methanol / 0.1 M hydrochloric acid (3+1) produced varying results. Compared with methanol / water extraction tested in IUPAC/AOAC intercomparison studies, as a rule acid extraction achieves a higher fumonisin content. Strong anion exchange columns are very effective for the analysis of untreated maize, such as maize grits or maize meal. This method guarantees satisfactory extract purification. In products with a high fat content (such as maize based snack products) or cornflakes, SAX columns were not particularly effective. For there immunoaffinity columns provide a highly selective extract purification and achieve a very pure chromatogram. Immunoaffinity columns were also used on cornflakes as they achieved better results than the SAX columns.