Peter M. Scott

Mycotoxins in infant cereal foods from the Canadian retail market

Three hundred and sixty-three samples of cereal-based infant foods were collected from the Canadian retail marketplace over 3 years. The samples included oat-, barley-, soy-, and rice-based infant cereals, mixed-grain infant cereals, teething biscuits, creamed corn, and soy-based formulas. Samples were analysed for targeted mycotoxins (deoxynivalenol, nivalenol, HT-2 toxin, zearalenone, ochratoxin A, fumonisins B1 and B2, and five ergot alkaloids). Soy-based cereals (which usually contain corn) exhibited the highest incidences of deoxynivalenol (100%), zearalenone (46%) and fumonisins (75%). Overall, deoxynivalenol was the most frequently detected mycotoxin — it was detected in 63% of samples analysed. Survey results demonstrated the regular occurrence of multiple mycotoxins in cereal-based infant foods.

Mycotoxins in botanicals and dried fruits: A review

Botanicals are used in many countries for medicinal and general health-promoting purposes. Numerous natural occurrences of mycotoxins in botanicals and dried fruits have been reported. Aflatoxins or ochratoxin A (OTA) have been found in botanicals such as ginseng, ginger, liquorice, turmeric, and kava-kava in the USA, Spain, Argentina, India, and some other countries, while fumonisins have been found in medicinal wild plants in South Africa and in herbal tea and medicinal plants in Turkey. Zearalenone was identified in ginseng root. Dried fruits can be contaminated with aflatoxins, OTA, kojic acid, and, occasionally, with patulin or zearalenone. One main area of concern is aflatoxins in dried figs; bright greenish yellow fluorescence under ultraviolet light is associated with aflatoxin contamination. OTA in dried vine fruits (raisins, sultanas, and currants) is another concern. There are also reports of aflatoxins in raisins and OTA in dried figs, apricots, dried plums (prunes), dates, and quince. Maximum permitted levels in the European Union include 4 µg kg−1 for total aflatoxins in dried fruit intended for direct consumption and 10 µg kg−1 for OTA in dried vine fruit. This review discusses the occurrence of mycotoxins in botanicals and dried fruits and analytical issues such as sampling, sample preparation, and methods for analysis. Fungal contamination of these products, the influence of sorting, storage, and processing, and prevention are also considered.

Fermentation of wort containing added ochratoxin A and fumonisins B1 and B2

Ochratoxin A (OA), fumonisin B1 (FB1) and fumonisin B2 (FB2) were added to wort at levels of 0.19, 0.95 and 0.95 micrograms/ml, respectively, and fermented for up to 8 days by three strains of Saccharomyces cerevisiae. Decreases of OA in the beer over this period were estimated from straight line slopes to be 2-13%. Losses of FB1 and FB2 were estimated to be 3-28% and 9-17% respectively. Some OA was taken up by the yeast, up to 21% in a detailed study with one strain. In contrast, uptake of fumonisins by yeast was negligible (< 1% FB1 and < 2% FB2). In control experiments, OA, FB1 and FB2 were found to be stable when added to yeast-free wort and kept for up to 8 days at 25 degrees C. In addition, spiking experiments with blank day 0-8 fermenting wort samples showed method recoveries averaging 87-91%. None of the mycotoxins was detected in control fermentations where they were not added to the wort.

Chronic Feeding Study of deoxynivalenol in B6C3F1 male and female mice

A 2 year feeding study was conducted with male and female B6C3F1 mice that consumed diets containing 0, 1, 5, or 10 ppm deoxynivalenol (DON). Survivability was good and, while the test animals gained less weight with increasing levels of DON in the diet, there were no consistent toxic manifestations associated with DON consumption. There was some evidence for an increase in serum IgA and IgG in females, and there were sporadic changes noted in the clinical chemistry and hematology parameters conducted at the terminal sacrifice. However, these changes were not considered to be biologically significant. The pathology results provided statistically significant dose-related evidence for a decrease in liver preneoplastic and neoplastic lesions as the dose level of DON increased. This negative trend probably results from the known positive correlation between body weight and the appearance of spontaneous hepatic neoplasms in this strain of mouse.

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 FB1. 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).

Multi‐year monitoring of Canadian grains and grain‐based foods for trichothecenes and zearalenone

Monitoring of Canadian grain crops and foods by the Health Protection Branch for deoxynivalenol (DON, vomitoxin) has been undertaken every year since 1980, when it was found in Ontario soft wheat for the first time (in the 1979 and 1980 crops). Contamination of this wheat crop has varied, with 22-100% incidences in all but 1 year and up to 0.75 micrograms/g for the annual means of positive samples. The Canadian guideline for DON is 2 micrograms/g in uncleaned soft wheat. Western Canadian hard wheat had < 10% incidence of DON in 7 crop years but 11-43% of samples analysed in 10 other years were positive. Wheat foods, including imports, have shown 9-90% incidences with annual mean levels of 0.07-0.58 micrograms/g in positive samples. Consistently high contamination of Ontario corn has been observed (13-100% annual incidences and annual means of positives 0.16-1.4 micrograms/g). Other trichothecenes, namely nivalenol and HT-2 toxin, have been found infrequently in Canadian grains. New analyses of Canadian and imported beers showed low ng/ml levels of DON. Grains destined for food use and corn foods have been analysed for zearalenone from 1986 to 1993. The most contaminated crop was Ontario; annual mean levels in positive samples ranged from 23 to 215 ng/g. Zearalenone has been detected infrequently in wheat, barley and soybeans (< 75 ng/g).

Liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry of the Alternaria mycotoxins alternariol and alternariol monomethyl ether in fruit juices and beverages

Alternariol (AOH) and alternariol monomethyl ether (AME) are among the main mycotoxins formed in apples and other fruits infected by Alternaria alternata. For determination of AOH and AME by LC, apple juice and other fruit beverages were cleaned up on C18 and aminopropyl solid-phase extraction columns. Positive and negative ion mass spectra of AOH and AME under electrospray (ESI) and atmospheric pressure chemical ionization (APCI) conditions were obtained. Collision-induced dissociation of the [M+H]+ and [M-H]- ions for both compounds were also studied. The phenolic anions of both compounds are more stable with less fragmentation. In quantitative analysis, negative ion detection also offers lower background and better sensitivity. Sensitive LC-MS and LC-MS-MS confirmatory procedures based on APCI with negative ion detection were applied to confirm the natural occurrence of AOH in nine samples of apple juice and in single samples of some other clear fruit beverages--grape juice, cranberry nectar, raspberry juice, red wine, and prune nectar (which also contained 1.4 ng AME/ml)--at levels of up to 6 ng AOH/ml. Electrospray LC-MS-MS with negative ion detection and in multiple reaction monitoring mode offers higher sensitivity and specificity. Absolute detection was better than 4 pg per injection for both compounds.

Quantitative determination of ochratoxin A by liquid chromatography/electrospray tandem mass spectrometry.

Mass spectrometry of ochratoxin A (OTA) and B (OTB) under electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) was studied. ESI offers higher sensitivities and less fragmentation than APCI. A sensitive LC/MS/MS method for the determination of ochratoxin A (OTA) in human plasma samples was developed. The absolute minimum detection limit was around 10-20 pg per injection, corresponding to 0.5 ppb in an injection equivalent to 20-40microg of human plasma. Ochratoxin B (OTB) was used as an internal standard and its absence in real-life samples was carefully checked before samples were spiked with the internal standard. It was found that these two ochratoxins are susceptible to sodium adduct formation. Fragment ions from the [M + H](+) and [M + Na](+) ions of both OTA and OTB were monitored in the multiple reaction monitoring mode. Three quantitative approaches, standard addition method, internal standard method (using ochratoxin B as an internal standard) and external standard method, were compared in the analysis of human blood plasma. Results from the mass spectrometric method were comparable to those from a conventional LC/fluorescence method. The LC/MS/MS method was also applied to the analysis of contaminated coffee samples.

Biomarkers of human exposure to ochratoxin A

Low blood serum/plasma concentrations of ochratoxin A (OTA) have been reported for healthy persons in more than 20 countries. Epidemiology studies in Bulgaria, Romania, Spain, the Czech Republic, Turkey, Italy, Egypt, Algeria and Tunisia have found significantly higher serum or plasma levels of OTA in patients with certain kidney disorders compared to healthy people, although the association may not be a causal one. Regional variations within one country, seasonal differences and variation within one person were found in some studies. Correlations with age and gender have not usually been detected. Detection limits using liquid chromatographic methods are about 0.02–0.1 ng ml−1 plasma/serum so that incidences of positives often are 50–100%, reflecting widespread and continuous exposure of humans to OTA. In a study in the UK, OTA in urine was found to be a better indicator of OTA consumption than OTA in plasma. Nevertheless, blood plasma concentrations have been widely used to estimate dietary intake of OTA, using equations relating it with plasma concentration, plasma clearance and bioavailability. A further source of human exposure is airborne dust. OTA has been detected in human milk in several countries and comparisons with serum/blood levels have been made in Germany and Sweden.

Mycotoxins in breakfast cereals from the Canadian retail market: A 3-year survey

One hundred and fifty-six samples of breakfast cereals were collected from the Canadian retail marketplace over a 3-year period. The samples were analysed for the mycotoxins deoxynivalenol, nivalenol, HT-2 toxin, zearalenone, ochratoxin A, and fumonisins B1 and B2 to contribute to dietary exposure estimates in support of the development of Canadian guidelines for selected mycotoxins in foods. The samples included corn-, oat-, wheat- and rice-based cereals, as well as mixed-grain cereals, and were primarily from North American processors. Overall, deoxynivalenol was the most frequently detected mycotoxin  — it was detected in over 40% of all samples analysed. Fumonisins and ochratoxin A were each detected in over 30% of all samples. Zearalenone was detected in over 20% of all samples. Nivalenol and HT-2 toxin were each detected in only one sample. The survey clearly demonstrated regular occurrence of low levels of multiple mycotoxins in breakfast cereals on the Canadian market.