Mycotoxins produced by Fusarium proliferatum and F. pseudonygamai on maize, sorghum and pearl millet grains in vitro.

Maize (Zea mays), sorghum (Sorghum bicolor) and pearl millet (Pennisetum glaucum) are basic staple foods for many rural or poorer communities. These crops are susceptible to plant diseases caused by multiple species of Fusarium, some of which also produce mycotoxins, including fumonisins and moniliformin that are detrimental to both humans and domesticated animals. Eighteen potentially toxigenic Fusarium strains were isolated from maize (n = 10), sorghum (n = 7) and pearl millet (n = 1) growing in the same field in Nigeria. The 17 strains from maize and sorghum were all F. proliferatum and the one strain from pearl millet was F. pseudonygamai. Under conducive conditions, the 17 F. proliferatum strains produced fumonisins, 11 in relatively large quantities (700-17,000 mg total fumonisins, i.e., FB1 + FB2 + FB3/kg culture material), and six at <45 mg/kg. Ten F. proliferatum strains produced >100 mg of moniliformin per kg culture material with a maximum of 8900 mg/kg culture material. All strains could use all grains for growth and toxin production, regardless of the host from which they were isolated. Isolates varied in the amount of toxin produced on each substrate, with toxin production a property of the strain and not the host from which the strain was recovered. However, the extent to which a toxin-producing phenotype could be altered by the grain on which the fungus was grown is consistent with subtle genetic × environment interactions that require a larger data set than the one presented here to rigorously identify. In conclusion, there is significant variation in the ability of strains of F. proliferatum to produce fumonisins and moniliformin on maize, sorghum and millet. If the amount of toxin produced on the various grains in this study reflects real-world settings, e.g., poor storage, then the consumers of these contaminated grains could be exposed to mycotoxin levels that greatly exceed the tolerable daily intakes.

The Mycotox Charter: Increasing Awareness of, and Concerted Action for, Minimizing Mycotoxin Exposure Worldwide.

Mycotoxins are major food contaminants affecting global food security, especially in low and middle-income countries. The European Union (EU) funded project, MycoKey, focuses on “Integrated and innovative key actions for mycotoxin management in the food and feed chains” and the right to safe food through mycotoxin management strategies and regulation, which are fundamental to minimizing the unequal access to safe and sufficient food worldwide. As part of the MycoKey project, a Mycotoxin Charter (charter.mycokey.eu) was launched to share the need for global harmonization of mycotoxin legislation and policies and to minimize human and animal exposure worldwide, with particular attention to less developed countries that lack effective legislation. This document is in response to a demand that has built through previous European Framework Projects—MycoGlobe and MycoRed—in the previous decade to control and reduce mycotoxin contamination worldwide. All suppliers, participants and beneficiaries of the food supply chain, for example, farmers, consumers, stakeholders, researchers, members of civil society and government and so forth, are invited to sign this charter and to support this initiative.

Mycotoxin Crises: Fit-for-Purpose Analytical Responses in the Developing World.

In developed market economies, control of mycotoxin exposure in the general population is achieved by legislated regulations governing maximum permitted levels. Such regulations are widely enforced to prevent outbreaks of overt mycotoxicoses. In developing countries, particularly in Africa, the situation is reversed, and individual mycotoxin exposures can be high, especially in rural communities reliant on subsistence or small-holder farming and local markets. Besides the effects of chronic mycotoxin exposure, Africa in recent years has experienced outbreaks of acute toxicity, such as aflatoxicosis. Recognizing and handling mycotoxin-induced health crises requires a range of responses, many of which rely on the provision and availability of fit-for-purpose analytical methods. Although regional laboratories may be able to provide support, rapid responses require in-field test kits reliant on antibody technologies. The future development of aptamers into test systems may be an important component of these analytical responses, as they provide important advantages in terms of stability, shelf-life, and low production costs.

Traditionally Processed Beverages in Africa: A Review of the Mycotoxin Occurrence Patterns and Exposure Assessment.

African traditional beverages are widely consumed food-grade liquids processed from single or mixed grains (mostly cereals) by simple food processing techniques, of which fermentation tops the list. These beverages are very diverse in composition and nutritional value and are specific to different cultures and countries. The grains from which home-processed traditional beverages are made across Africa are often heavily contaminated with multiple mycotoxins due to poor agricultural, handling, and storage practices that characterize the region. In the literature, there are many reports on the spectrum and quantities of mycotoxins in crops utilized in traditional beverage processing, however, few studies have analyzed mycotoxins in the beverages themselves. The available reports on mycotoxins in African traditional beverages are mainly centered on the finished products with little information on the process chain (raw material to final product), fate of the different mycotoxins during processing, and exposure estimates for consumers. Regulations targeting these local beverages are not in place despite the heavy occurrence of mycotoxins in their raw materials and the high consumption levels of the products in many homes. This paper therefore comprehensively discusses for the 1st time the available data on the wide variety of African traditional beverages, the mycotoxins that contaminate the beverages and their raw materials, exposure estimates, and possible consequent effects. Mycotoxin control options and future directions for mycotoxin research in beverage production are also highlighted.

Current Status of Mycotoxin Analysis: A Critical Review.

It is over 50 years since the discovery of aflatoxins focused the attention of food safety specialists on fungal toxins in the feed and food supply. Since then, analysis of this important group of natural contaminants has advanced in parallel with general developments in analytical science, and current MS methods are capable of simultaneously analyzing hundreds of compounds, including mycotoxins, pesticides, and drugs. This profusion of data may advance our understanding of human exposure, yet constitutes an interpretive challenge to toxicologists and food safety regulators. Despite these advances in analytical science, the basic problem of the extreme heterogeneity of mycotoxin contamination, although now well understood, cannot be circumvented. The real health challenges posed by mycotoxin exposure occur in the developing world, especially among small-scale and subsistence farmers. Addressing these problems requires innovative approaches in which analytical science must also play a role in providing suitable out-of-laboratory analytical techniques.

Relative severity of fumonisin contamination of cereal crops in West Africa.

Traditional and improved varieties of maize, pearl millet and sorghum were planted by small-scale farmers under the direction of the International Institute for Tropical Agriculture in two Nigerian agro-ecological zones: the Sudan Savanna and the Northern Guinea Savanna. Samples were collected for the determination of Fusarium infection and fumonisin (B1, B2 and B3) contamination. A previous paper reported Aspergillus infection and aflatoxin contamination of these samples. Fusarium infection levels, measured by per cent kernels infected, were modest with mean levels for the above cereals of 16% ± 11% (SD), 12% ± 7% and 13% ± 16%, respectively. However, the Fusarium species recovered from maize were predominantly the fumonisin producers F. verticillioides and F. proliferatum, together making an infection rate of 15% ± 10%, whereas these species were present to a limited extent only in the other two cereals, 1% ± 1% for pearl millet and 2% ± 6% for sorghum. Fumonisin contamination was variable but reflected the diversity of Fusarium producers in these three cereals. Mean levels were 228 ± 579 µg kg(-1) (range < 5-2860 µg kg(-1)) for maize, 18 ± 7 µg kg(-1) (range = 6-29 µg kg(-1)) for pearl millet and 131 ± 270 µg kg(-1) (range < 5-1340 µg kg(-1)) for sorghum. Together with previous results on aflatoxin, this study confirmed the co-occurrence of aflatoxins and fumonisins in maize as well as in the traditional African cereals, millet and sorghum (89% co-occurrence across all three cereals). The low fumonisin levels may be ascribed to the use of good agricultural practices. Of the Fusarium species present, those in maize consisted mainly of fumonisin producers, the opposite of what was observed in pearl millet and sorghum. It is concluded that replacement of maize by pearl millet and sorghum could improve food safety with regards to aflatoxin B and fumonisin B exposure.

Development and evaluation of a sensitive mycotoxin risk assessment model (MYCORAM).

The differential risk of exposure to fumonisin (FB), deoxynivalenol (DON), and zearalenone (ZEA) mycotoxins to the South African population, residing in the nine Provinces was assessed during a cross-sectional grain consumer survey. The relative per capita maize intake (g/day) was stratified by gender, ethnicity, and Province and the probable daily intake (PDI) for each mycotoxin (ng/kg body weight/day) calculated utilizing SPECIAL and SUPER dry milled maize fractions representing different exposure scenarios. Men consumed on an average more maize (173 g/day) than women (142 g/day) whereas the black African ethnic group had the highest intake (279 g/day) followed by the Colored group (169 g/day) with the Asian/Indian and White groups consuming lower quantities of 101 and 80 g/day, respectively. The estimated mean PDIs for the various subgroups and Provinces, utilizing the different dry milled maize fractions, were below the provisional maximum tolerable daily intake (PMTDI) for each mycotoxin. A distinct and more sensitive mycotoxin risk assessment model (MYCORAM) for exposure, stratified by Province and ethnicity were developed utilizing specific maize intake increments (g/kg body weight/day) that provides information on the percentage of the population exposed above the PMTDI for each mycotoxin. Evaluation of the MYCORAM utilizing commercial and EXPERIMENTALLY DERIVED: SPECIAL milling fractions, containing predefined mycotoxins levels, predicts the percentage of maize consumers exposed above the respective PMTDI. Safety modeling using the MYCORAM could also predict a maximum tolerated level adequate to safeguard all South African maize consumers including the most vulnerable groups.

Mycological analysis and multimycotoxins in maize from rural subsistence farmers in the former Transkei, South Africa.

Maize harvested in the Centane region of the former Transkei, Eastern Cape Province, South Africa, by subsistence farmers has been shown over many seasons to be contaminated with fumonisin mycotoxins. However, there are limited data on the presence of other mycotoxins. Two multimycotoxin LC-MS/MS methods were applied to good and moldy maize samples, as separated by the farmers themselves from the 2011 harvest. One method involved extract cleanup on multitoxin immunoaffinity columns before LC-MS/MS analysis for aflatoxins, fumonisins, deoxynivalenol (DON), zearalenone (ZEN), and T-2 and HT-2 toxins. The other method was based on a "dilute-and-shoot" approach for the above mycotoxins and a wide range of other fungal secondary metabolites. Both methods showed high incidences of fumonisins B1 and B2 (FB1 and FB2) in good maize (100% for both by the first method, means were 2083 and 927 µg/kg for the two analogues; 93% for both by the second method, positive means of 2764 and 1050 µg/kg, respectively). All samples of moldy maize were contaminated (mean FB1 of 27.64 and 35.98 mg/kg, respectively; mean FB2 of 10.58 and 14.14 mg/kg, respectively). Comparison of the two methods for FB1 and FB2 over the entire range of samples gave R(2) values 0.9144 and 0.8859, respectively. Low levels of DON were found by both methods (positive means of 12 and 4.7 µg/kg in good maize, respectively, and of 14 and 5.8 µg/kg in moldy maize, respectively). ZEN was determined with positive means of 108 and 25 µg/kg in good maize, respectively, and of 111 and 135 µg/kg in moldy maize, respectively. No aflatoxins, OTA, or T-2 or HT-2 toxins were detected. A wide range of other Fusarium , Aspergillus , Alternaria , and Penicillium mycotoxins and secondary metabolites were determined.

Multiple mycotoxin exposure determined by urinary biomarkers in rural subsistence farmers in the former Transkei, South Africa.

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxin LC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancer region, Transkei, South Africa. Fifty-three female participants donated part of their maize-based evening meal and first void morning urine, which was analysed both with sample clean-up (single and multi-biomarker) and by a 'dilute-and-shoot' multi-biomarker method. Results were corrected for recovery with LOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean±standard deviation 0.342±0.466 ng/mg creatinine) and deoxynivalenol (100%; mean 20.4±49.4 ng/mg creatinine) after hydrolysis with ß-glucuronidase. The multi-biomarker 'dilute-and-shoot' method indicated deoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with ß-glucuronidase and immunoaffinity clean-up determined zearalenone (100%; 0.529±1.60 ng/mg creatinine), FB1 (96%; 1.52±2.17 ng/mg creatinine), α-zearalenol (92%; 0.614±1.91 ng/mg creatinine), deoxynivalenol (87%; 11.3±27.1 ng/mg creatinine), ß-zearalenol (75%; 0.702±2.95 ng/mg creatinine) and ochratoxin A (98%; 0.041±0.086 ng/mg creatinine). These demonstrate the value of multi-biomarker methods in measuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinary deoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei.

Fumonisin B1 and risk of hepatocellular carcinoma in two Chinese cohorts.

Fumonisin B1 (FB1), a mycotoxin that contaminates corn in certain climates, has been demonstrated to cause hepatocellular cancer (HCC) in animal models. Whether a relationship between FB1 and HCC exists in humans is not known. To examine the hypothesis, we conducted case-control studies nested within two large cohorts in China; the Haimen City Cohort and the General Population Study of the Nutritional Intervention Trials cohort in Linxian. In the Haimen City Cohort, nail FB1 levels were determined in 271 HCC cases and 280 controls. In the General Population Nutritional Intervention Trial, nail FB1 levels were determined in 72 HCC cases and 147 controls. In each population, odds ratios and 95% confidence intervals (95%CI) from logistic regression models estimated the association between measurable FB1 and HCC, adjusting for hepatitis B virus infection and other factors. A meta-analysis that included both populations was also conducted. The analysis revealed no statistically significant association between FB1 and HCC in either Haimen City (OR=1.10, 95%CI=0.64-1.89) or in Linxian (OR=1.47, 95%CI=0.70-3.07). Similarly, the pooled meta-analysis showed no statistically significant association between FB1 exposure and HCC (OR=1.22, 95%CI=0.79-1.89). These findings, although somewhat preliminary, do not support an associated between FB1 and HCC.

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.

Aflatoxins and growth impairment: a review.

Aflatoxins, fungal toxins produced by Aspergillus flavus and Aspergillus parasiticus in a variety of food crops, are well known as potent human hepatocarcinogens. Relatively less highlighted in the literature is the association between aflatoxin and growth impairment in children. Foodborne aflatoxin exposure, especially through maize and groundnuts, is common in much of Africa and Asia--areas where childhood stunting and underweight are also common, due to a variety of possibly interacting factors such as enteric diseases, socioeconomic status, and suboptimal nutrition. The effects of aflatoxin on growth impairment in animals and human children are reviewed, including studies that assess aflatoxin exposure in utero and through breastfeeding. Childhood weaning diets in various regions of the world are briefly discussed. This review suggests that aflatoxin exposure and its association with growth impairment in children could contribute a significant public health burden in less developed countries.

Fumonisin B1 as a urinary biomarker of exposure in a maize intervention study among South African subsistence farmers.

BACKGROUND: The consumption of maize highly contaminated with carcinogenic fumonisins has been linked to high oesophageal cancer rates. The aim of this study was to validate a urinary fumonisin B1 (UFB1) biomarker as a measure of fumonisin exposure and to investigate the reduction in exposure following a simple and culturally acceptable intervention. METHODS: At baseline home-grown maize, maize-based porridge, and first-void urine samples were collected from female participants (n=22), following their traditional food practices in Centane, South Africa. During intervention the participants were trained to recognize and remove visibly infected kernels, and to wash the remaining kernels. Participants consumed the porridge prepared from the sorted and washed maize on each day of the two-day intervention. Porridge, maize, and urine samples were collected for FB1 analyses. RESULTS: The geometric mean (95% confidence interval) for FB1 exposure based on porridge (dry weight) consumption at baseline and following intervention was 4.84 (2.87-8.14) and 1.87 (1.40-2.51) µg FB1/kg body weight/day, respectively, (62% reduction, P<0.05). UFB1C, UFB1 normalized for creatinine, was reduced from 470 (295-750) at baseline to 279 (202-386) pg/mg creatinine following intervention (41% reduction, P=0.06). The UFB1C biomarker was positively correlated with FB1 intake at the individual level (r=0.4972, P<0.01). Urinary excretion of FB1 was estimated to be 0.075% (0.054%-0.104%) of the FB1 intake. CONCLUSION: UFB1 reflects individual FB1 exposure and thus represents a valuable biomarker for future fumonisin risk assessment. IMPACT: The simple intervention method, hand sorting and washing, could positively impact on food safety and health in communities exposed to fumonisins.

Determination of aflatoxin in processed dried cassava root: validation of a new analytical method for cassava flour.

A new method that uses HPLC with a photochemical reactor for enhanced detection was developed and validated for the determination of aflatoxins in cassava flour. Samples were spiked with a mixture of four aflatoxins at 5, 10, and 20 microg/kg mixed with either 1 or 5 g NaCI and extracted with methanol-water (80 + 20, v/v) by shaking for 10 or 30 min. An immunoaffinity column was used for cleanup. HPLC with postcolumn derivatization, for enhancement of aflatoxin fluorescence, and fluorescence determination were used for quantitation of the toxin concentration. The method was validated for recovery, linearity, and precision at the three concentrations tested. Recovery ranges were 52-70, 69-85, and 80-89% for the spiking levels of 5.0, 10.0, and 20.0 microg/kg, respectively. It appears that the amount of salt (NaCl) and the shaking time are critical factors in this method; optimal performance was obtained when 1 g salt was used and the shaking time was 10 min. The good linearity and precision of the method allowed baseline separation from interferences, e.g., coumarins.

Preliminary exposure assessment of deoxynivalenol and patulin in South Africa.

Deoxynivalenol (DON) and patulin (PAT) are mycotoxins widely regulated internationally. DON is frequently found in cereals, whereas PAT is commonly found in apple juices. A survey of South African commercial products was conducted on DON levels in maize meal and wheat flours, and on PAT levels in apple juices. DON levels in 23 wheat flour samples (mean of 16 positives, 29 µg/kg) were equal to or below 100 µg/kg and in wheat consumers contributed 6-13% of the provisional maximum tolerable daily intake (PMTDI; 1 µg/kg body weight per day) for DON set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Sixteen of 18 maize meal samples were contaminated, with a mean for positive samples of 294 µg/kg, and the probable daily intakes ranged from 3.67 µg/kg body weight per day in rural infants to 1.39 µg/kg body weight per day in urban adults. PAT levels in 20 of 30 apple-juice samples were below the detection level of 10 µg/l. Mean of positive samples was 210 µg/l, with three samples exceeding the South African legal limit of 50 µg/l and the highest level (1,650 µg/l) showing the possibility of a brief but high exposure of 37 µg/kg body weight per day (or 9,250% of the JECFA PMTDI of 0.4 µg/kg body weight per day) in young children.

Aflatoxin analysis at the beginning of the twenty-first century.

Aflatoxin mycotoxins were first described in the early 1960s as important fungal toxins, which contaminate many different human foods and animal feeds. Accurate and sensitive determination of these carcinogenic compounds immediately became an important requirement to meet food safety concerns and new official legislated regulations. For these reasons, analytical methods for aflatoxins continued to develop over the decades, reflecting advances in analytical chemistry. Currently, a wide range of methods are available to analytical scientists, ranging from newly described multi-toxin liquid chromatography tandem mass spectrometry to rapid methods based on immunological principles. These latter methods can provide quantitative outputs or a simple rapid determination of contamination level above or below a pre-determined cutoff value. The newest official methods as validated by Association of Official Analytical Chemists International or Comité Européen de Normalisation rely on immunoaffinity column clean-up of conventional extracts, followed by high-performance liquid chromatography separation of the analogues with detection based on natural fluorescence or the fluorescence generated by various derivatisation methods. In selecting from this range of available methods, the analytical chemist must decide on the requirements of the analysis such that the method chosen is 'fit for purpose'.

Determination of fumonisins in maize by HPLC with ultraviolet detection of o-phthaldialdehyde derivatives.

Fumonisins are mycotoxins that are produced by various Fusarium species and occur naturally in maize and maize-based foods. Fumonisins are carcinogenic, causing liver cancer in rats, and are associated with oesophageal cancer and neural tube defects in humans. Analytical methods for individual fumonisin analogues in maize rely on reversed-phase high-performance liquid chromatographic (HPLC) separation after suitable extraction and clean-up. As fumonisins lack a useful chromophore or fluorophore, HPLC detection is achieved by suitable derivatization and sensitive, specific fluorescence detection. A widely used and validated method involves extract clean-up on strong anion exchange solid phase extraction cartridges and pre-column derivatization with o-phthaldialdehyde (OPA). However, many laboratories requiring infrequent fumonisin analysis are only equipped with HPLC with ultraviolet (UV) detection. A HPLC system equipped with both UV and fluorescence detectors connected in series was used to determine the extent to which UV offers an alternative to fluorescence detection in the above analytical method. Comparison of the detection systems using fumonisin standards indicated that fluorescence is about 20-times more sensitive than UV. Analysis of maize samples with differing fumonisin contamination levels indicated that, at fumonisin B1 levels above 1,000 µg/kg, the two detection systems were comparable and gave repeatabilities equal or less than 10% on six replicate analyses. Although a sensitive fumonisin analysis requires fluorescence detection, UV may offer an alternative in certain circumstances.

Risk assessment of aflatoxins in food in Africa.

Aflatoxins are secondary metabolites of the fungi Aspergillus flavus and A. parasiticus, occur widely on many staple foods and cause a broad range of detrimental health effects in animals and humans. As a consequence, maximum tolerated levels (MTLs) have been legislated in many countries. However, in developing countries where food safety compliance can be low and significant levels of the food supply are locally consumed by the producers or purchased at local markets, more comprehensive strategies are required. In this regard, risk analysis with its components of risk assessment, risk management and risk communication, is an important tool in dealing with food safety issues. Risk assessment for aflatoxin B(1) in Africa has been performed using the carcinogenic potency, established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and applying it to typical food products and consumption across the continent, to illustrate the significant health implications caused by the intake of high levels of contaminated foods. Highlighted in this assessment is the fact that even low levels of contamination, which might of themselves fall within legislated limits, can have serious health implications due to high levels of consumption, i.e. meeting a MTL does not of itself guarantee food safety. Recent developments have highlighted the growth retardation and immune suppression caused by aflatoxin exposure in human populations in west Africa. Using the limited data available on both these health effects, a first step has been taken to incorporate them into a risk assessment paradigm quantifying the risk of immunosuppression, malnutrition and stunting in children exposed to aflatoxins and highlighting again how excessive consumption of foods meeting MTLs can carry significant health risks.

Structure and natural occurrence of stereoisomers of the fumonisin B series mycotoxins.

1H and 13C NMR spectroscopy of both fumonisin B3 and B4, as well as high-performance liquid chromatography (HPLC) analysis of samples of fumonisin B3 used as standards, showed in each case the presence of two stereoisomers, which could not be separated by preparative chromatography. The 2,3-anti relative configuration for the two minor stereoisomers of fumonisin B3 and B4 was deduced from the NMR data, and their 2S,3R absolute configurations were established by application of Mosher's method using the fumonisin B3 sample. Samples of fumonisin B3 and B4 can contain between 10 and 40% of fumonisin B compounds of the 3-epi series. The 3-epi-FB3, determined by HPLC with fluorescence detection of the o-phthaldialdehyde derivative and confirmed by liquid chromatography-tandem mass spectrometry, was found to occur naturally in a range of maize samples at levels much lower than FB3 (< 20%). The identification of members of the 3-epi-fumonisin B series provides insight into the order and selectivity of steps in fumonisin biosynthesis.

Determination of patulin in apple juice: comparative evaluation of four analytical methods.

The performance of 4 purification methods for the analysis of patulin in apple juice was evaluated by high-performance liquid chromatography (HPLC). Samples were spiked with patulin at 10, 20, 50, 100, and 150 ppb (ng/mL) and extracted by one of 4 methods (3 solid-phase extraction and one liquid-liquid extraction), and then analyzed by HPLC-UV under the same isocratic conditions. The methods were validated for recovery, linearity, and precision at high and low concentrations. Recoveries were all >70% for spiking range 10-150 ppb. The relative standard deviation for repeatability was found to meet European Union Directive requirements. In addition, all the methods showed baseline separation from hydroxymethylfurfural.