Analysis of wines, grape juices and cranberry juices forAlternaria toxins.

Sixty six samples of red and white wine from Ontario (VQA), British Columbia (VQA), Québec ("vins artisanaux"), imported wines (from Italy, South America and USA) and Canadian and US grape and cranberry juices were analysed for theAlternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME). After cleanup on aminopropyl SPE columns, AOH and AME were initially determined by reversed phase LC with UV detection. Positive sample extracts were re-analysed by LC-tandem negative ion electrospray mass spectrometry (MS/MS) in multiple reaction mode. Overall mean method recoveries measured by LC-UV were 93% for AOH and 81% for AME. Limits of detection in wine (and juice) by LC-UV for AOH were 0.8 (0.4) ng/ml and for AME were 0.5 (0.4) ng/ml; they were below 0.01 ng/ml by LC-MS/MS. As determined by LC-MS/MS, AOH was found in 13/17 Canadian red wines at levels of 0.03 to 5.02 ng/ml and in 7/7 imported red wines at 0.27-19.4 ng/ml, usually accompanied by lower concentrations of AME. Red grape juices (5 positive/10 samples) contained only sub ng/ml levels of AOH or AME except for one sample (39 ng AME/ml). White wines (3/23 samples), white grape juices (0/4 samples) and cranberry juices (1/5 samples) contained little AOH/AME (≤1.5 ng/ml).

Analysis of Ginkgo biloba for the presence of ginkgotoxin and ginkgotoxin 5'-glucoside.

Hot water extracts of Ginkgo biloba seeds were analyzed for the presence of ginkgotoxin (4'-O-methylpyridoxine) by reversed-phase liquid chromatography (LC) using methanol-0.05M KH2PO4 (1 + 9, v/v) adjusted to pH 3 as mobile phase. Detection was by fluorescence (excitation 280 nm, emission 370 nm). A straight line calibration curve was obtained for the 10-100 ng injected. After addition of beta-glucosidase (37 degrees C/h), an earlier eluting peak disappeared and the ginkgotoxin peak increased. The identity of the ginkgotoxin was confirmed by LC/MS and LC/MS/MS. LC/MS/MS also confirmed the 5'-glucoside by comparison with the 3-glucoside. This is the first identification of a glucoside of ginkgotoxin in Ginkgo biloba. An unknown compound of MW 267 also observed in the Ginkgo biloba seed extract was shown not to be 3,5'-diacetylginkgotoxin by its different LC retention time. Extraction of ground Ginkgo biloba seeds with boiling water in a Soxhlet for 2 x 2 h yielded a total of 179 microg/g of free ginkgotoxin. The concentration in powder from Ginkgo biloba capsules was several times lower than this (17-64 microg/g) in 3 samples but higher in another (457 microg/g). Canned ginkgo seeds (white nuts) contained no detectable free ginkgotoxin but the glucoside was present. Different extraction times were studied: 0.5 h gave only 52 microg/g free ginkgotoxin in the ginkgo seeds. However, boiling an extract for 4 h showed about 15% loss of ginkgotoxin and its glucoside.

Studies on extraction of fumonisins from rice, corn-based foods and beans.

Different solvent mixtures were examined for extraction of fumonisins from various naturally contaminated and spiked foods and foodstuffs: rough rice, retail rice, rice flour, white corn flour, corn meal, corn starch, corn flakes, tortilla/corn chips, white bean flour, white beans, mung beans, adzuki beans and infant cereals. Most of the naturally contaminated samples were analyzed using the extraction solvent mixtures methanol-acetonitrile-water (25:25:50) (solvent A) and methanol-water (75:25 or 80:20) (solvents B, BB); some were extracted with 0.1 M sodium hydrogen phosphate-acetonitrile (1:1, adjusted to pH 3.0 with o-phosphoric acid) (solvent C) and methanol-0.025 M borate buffer (3:1, adjusted to pH 9.2 with 1 N sodium hydroxide) (solvent D). A 1-ml SAX solid phase extraction column was used for the cleanup in all cases except for infant cereals, for which immunoaffinity chromatography was used; fumonisin concentrations were determined by liquid chromatography.Solvent A gave slightly better extraction of fumonisins from one of two samples of naturally contaminated rough rice than solvent B (fumonisin B1: 4080 ng/g versus 3150 ng/g; fumonisin B2:1100 ng/ g versus 922 ng/g) and much better extraction than solvent C (1210 ng/g fumonisin B1 and 315 ng/g fumonisin B2) or solvent D (372 ng/ g fumonisin B1 and 191 ng/g fumonisin B2). However, spike recoveries on a similar rice naturally contaminated at a lower level were only in the 43-53% range (solvent A). Recovery of fumonisins was very poor from spiked white rice flour but satisfactory from other rice foods.Solvent A similarly gave slightly better extraction of fumonisins from a sample of naturally contaminated white corn flour than solvent B (fumonisin B1 1260 ng/g versus 931 ng/g; fumonisin B2: 511 ng/g versus 447 ng/g ) and better extraction than solvents C and D. Solvent A was also a better solvent for extraction of fumonisins from naturally contaminated tortilla chips and infant cereals. Study of naturally contaminated corn starch was confounded by instability of fumonisins in this food. Recovery of fumonisins from spiked corn meal, tortilla chips, corn flakes, various types of beans and infant cereals with solvent A and/or solvent B (or BB) was satisfactory.

Fumonisin levels in Uruguayan corn products.

A survey was conducted to evaluate fumonisins FB1 and FB2 in Uruguayan corn products. Sixty-four samples of different local brands were purchased from retail stores during a 15-month period and analyzed for FB1 and FB2 by methanol-water extraction, cleanup with a 1 mL. strong-anion-exchange solid-phase extraction column, and liquid chromatography with o-pthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. Contamination levels for FB1 varied from 50 ng/g (detection limit) to 6342 ng/g. Values were highest in feed samples (up to 6342 ng/g), unprocessed corn kernel (up to 3688 ng/g), and milled products, which included polenta (up to 427 ng/g). They were lowest in processed corn kernel (up to 155 ng/g) and snacks (up to 314 ng/g). FB2 was determined in one-fourth of the total samples and detected at trace levels in only one feed sample. The data demonstrated the natural occurrence of fumonisins in corn products in Uruguay. Feed and polenta that contain fumonisins could be of concern because they are consumed in large amounts and are often the main nutrient source in Uruguay.

Evaluation of enzyme-linked immunosorbent assay for analysis of beer for fumonisins.

A recently developed sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was applied to the determination of fumonisins in beer. Intra-assay and inter-assay recoveries averaged 98.7-102.8% at added fumonisin B1 (FB1) levels of 0.5-50 ng/ml beer, and coefficients of variation were 2.8-4.4 and 4.7-8.6%, respectively. Cross-reactivity of fumonisin B2 (FB2) compared with FB1 averaged 67% in beer. Two experiments were carried out to compare ELISA with liquid chromatography (LC) for determination of fumonisins in beer. In the first experiment, 19 samples (five previously known positive, nine other samples and five spiked samples) were passed through commercial immunoaffinity columns (ICs) and analysed by LC before conducting blind ELISA determinations on the extracts and beers directly. The known positive beers and extracts were used as blind duplicates. The second comparative experiment screened 46 beer samples by ELISA and then 22 positive and three of the negative samples were analysed by LC; the highest level found was 64.3 ng total fumonisins/ml measured by LC (24.7 ng/ml by ELISA). Regression analyses showed good correlation between ELISA and LC in the first experiment but low level interferences (equivalent to up to 5.35 ng fumonisin/ml) were observed by ELISA in the IC extracts. Five of nine beers negative by LC showed < 1 ng/ml ELISA responses on direct beer analysis. The second comparative experiment indicated underestimation by ELISA. However, there were two samples which tested positive by ELISA (0.2 ng/ml) but were found negative by LC (results close to the detection limits of both methods, which were 0.1 or 0.2 ng/ml by ELISA and 0.1-0.15 ng each fumonisin/ml by LC). There were no false negatives. It is concluded that ELISA has considerable value in rapid screening of beer directly for fumonisins.

Determination of aflatoxins in beer.

Aflatoxins B1, B2, G1, and G2 were determined at parts-per-trillion levels in beer by immunoaffinity column cleanup and reversed-phase liquid chromatography (LC) with fluorescence detection after trifluoroacetic acid derivatization. Silanized vials were necessary for the evaporation step in order to obtain good recoveries of aflatoxins from spiked beer samples. Recoveries averaged 90-104%, 94%, 84-87%, and 89% for aflatoxins B1, B2, G1, and G2, respectively, at levels of 9.7-133 ng B1, 46 ng B2, 35-140 ng G1, and 41 ng G2/L. Detection limits were 19-20 ng/L for aflatoxins B1 and G1 and 15-16 ng/L for aflatoxins B2 and G2 (signal-to-noise ratio = 3:1) obtained by using an excitation wavelength of 360 nm; at 340 nm these detection limits were lowered to about 2 ng/L. Analysis of 24 beer samples, the majority from the United States and Mexico, showed natural contamination of one sample of Mexican beer at 49 ng B1/L when determined at 360 nm excitation, but reanalysis of 23 of the samples using 340 nm excitation indicated that an additional 4 Mexican samples and one Brazilian sample contained aflatoxin B1 at low levels (< 10 ng/L).

Determination of hydrolysed fumonisin B1 in alkali-processed corn foods.

Treatment of fumonisin B1 (FB1)-contaminated corn with calcium hydroxide solution is known to cause large losses of FB1 and formation of the aminopentol AP1 by hydrolysis. Methodology was developed for determination of AP1 in foods manufactured from calcium hydroxide-processed corn. The ground food (tortilla chips, nacho chips, taco shells, or air-dried corn tortillas) was extracted with methanol-water (8:2) or methanol-acetonitrile-water (25:25:50), which also extracted FB1 and fumonisin B2 (FB2). Clean-up for fumonisin determination was carried out on a 1 ml strong anion exchange (SAX) solid phase extraction (SPE) column. The water wash from this column, containing AP1, was cleaned up on a 1 ml C-18 SPE column. AP1 and, separately, FB1 and FB2 were determined as their o-phthaldialdehyde-mercaptoethanol (OPA/MCE) and, in some cases, 4-fluoro-7-nitrobenzofurazan (NBD-F) derivatives by gradient reverse phase liquid chromatography with fluorescence detection. Recoveries of AP1, FB1 and FB2 from spiked samples were generally satisfactory, but FB1 and FB2 recoveries were low with some samples. Better recovery of FB1 with the methanol-acetonitrile-water (25:25:50) extraction solvent compared with methanol-water (8:2) was observed for naturally-contaminated samples. Detection limits were about 10 ng AP1 per g and 20 ng FB1 and FB2 per g with OPA/MCE derivatization, but there were interferences for FB2. Analysis of 31 samples of alkali-processed corn foods (including three known already to contain FB1) showed measurable levels of AP1 in nine samples, all < 100 ng/g and lower than the corresponding FB1 concentrations.

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.

Stability and problems in recovery of fumonisins added to corn-based foods.

Because the natural occurrence of fumonisins is so far known almost exclusively in corn, we have limited our investigations on their stability to corn-based foods. In these studies, distinction must be made between real losses, binding, and any matrix-related method problems. Fumonisins B1 (FB1) and B2 (FB2) were about 40% recovered when heated in corn meal at 190 degrees C, about 20-30% recovered when heated in moist corn meal at 190 degrees C, and completely unstable in corn meal at 220 degrees C. Average recoveries of FB1 and FB2 added to blank heated matrixes were 69-107% in control experiments. Baking corn meal muffins spiked with 2.5 micrograms FB1 and FB2/g corn meal at 220 degrees C also resulted in losses of fumonisins. Little or no fumonisins were recovered from corn bran flour when methanol-water (3 + 1) was used as extraction solvent. However, when methanol-borate buffer (pH 9.2) (3 + 1) was used, recoveries averaged 91 +/- 17 and 84 +/- 9%, respectively, for FB1 and FB2; and natural contamination of the corn bran flour with FB1 and FB2 at levels of 1.9 and 0.95 microgram/g, respectively, was revealed. Comparable recoveries were observed for 1 brand of a corn bran breakfast cereal, but the binding effect was not seen with a second brand, for which methanol-water (3 + 1) alone was a good extraction solvent. Recoveries of FB1 and FB2 from a mixed cereal for babies were only about 50% with either extraction solvent mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Minimal transmission of zearalenone to milk of dairy cows.

Milk and plasma levels of zearalenone (ZEN), alpha-zearalenol (alpha-ZEL), beta-zearalenol (beta-ZEL) and conjugated metabolites were determined after feeding lactating cows with ZEN. In those instances where ZEN and alpha- and beta-ZEL were detected in milk or plasma, they occurred only as conjugates hydrolysable by treatment with a mixture of beta-glucuronidase and aryl sulfatase. With studies where 50 or 165 mg was fed daily to three cows for 21 day periods, neither dosage showed the presence of ZEN or metabolites in either milk or plasma (detection limits: milk, 0.5 ng/ml, ZEN, alpha-ZEL; 1.5 ng/ml, beta-ZEL; plasma, 2-3 times higher). A dose of 544.5 mg zearalenone per day given to a single cow for 21 days yielded maximum concentrations of only 2.5 ng ZEN/ml and 3.0 ng alpha-ZEL/ml in the milk. In plasma, up to 3 ng ZEN/ml could be detected during the initial 4 days of treatment. At a dose of 1.8 g of zearalenone given over a one day feeding period, maximum milk levels of 4.0 ng ZEN/ml, 1.5 ng alpha-ZEL/ml, and 4.1 ng beta-ZEL/ml were observed during the initial 2 days; corresponding maximum levels after a one day dose of 6.0 g zearalenone were 6.1, 4.0 and 6.6 ng/ml milk on days 2-3. In plasma, peak ZEN concentrations (9 and 13 ng/ml at the lower and higher one-day doses, respectively) occurred 12 hr after initial dosing, and declined to negligible levels by days 5-7. Neither alpha- nor beta-ZEL were detected in plasma. Since measurable levels required very high oral doses of ZEN, milk would not normally pose a human health hazard as a result of feeding rations containing ZEN to lactating dairy cows.

Liquid chromatographic determination of zearalenone and alpha- and beta-zearalenols in milk.

Previous research has demonstrated transmission of zearalenone and alpha- and beta-zearalenols into the milk of cows and other animals. Since human intake of zearalenone and its metabolites via milk is an unknown factor in risk assessment of zearalenone and because appropriate methodology for their determination in milk is not available, a rapid and sensitive analytical method has been developed. Essentially, the method includes extraction with basic acetonitrile, acidification, partition into methylene chloride on a hydrophilic matrix, cleanup on an aminopropyl solid phase extraction column, and reverse-phase liquid chromatography with fluorescence detection. Recoveries from milk averaged 84% for zearalenone, 93% for alpha-zearalenol, and 90% for beta-zearalenol at spiking levels of 0.5 to 20 ng/mL. As little as 0.2 ng/mL of zearalenone and alpha-zearalenol and 2 ng/mL of beta-zearalenol can be detected in milk. These 3 compounds are stable in refrigerated milk for at least 2 weeks and in milk brought to boiling. Enzymes (beta-glucuronidase and aryl sulfatase) may be added to milk prior to extraction to hydrolyze any conjugates.

Production of moniliformin by Canadian isolates of Fusarium.

Twenty-eight Canadian isolates of Fusarium were tested for their ability to produce moniliformin in corn. Both F. moniliforme (2/6 isolates) and F. subglutinans (11/15 isolates) produced the mycotoxin, while F. graminearum did not. Field-corn inoculated with F. moniliforme M3783 was able to support production of both moniliformin and fusarin C.

Liquid chromatographic determination and stability of the Fusarium mycotoxin moniliformin in cereal grains.

Moniliformin is a mycotoxin produced by Fusarium subglutinans and other Fusarium species. A rapid, liquid chromatographic method for its determination in corn and wheat is described. Samples are extracted in acetonitrile-water (95 + 5); following defatting with n-hexane, an aliquot of the extract is evaporated and cleaned up on small C18 and neutral alumina columns successively. Reverse-phase liquid chromatography (LC) is conducted on a C18 column with 10 or 15% methanol or acetonitrile in aqueous ion-pair reagent as mobile phase, with detection by ultraviolet absorption at 229 and 254 nm. Average recoveries of moniliformin (potassium salt) added to ground corn and wheat at levels of 0.05-1.0 micrograms/g were 80% (n = 20) and 85% (n = 12), respectively, and the limit of detection was ca 0.01-0.18 micrograms/g, depending on LC conditions. Analysis of 24 samples of wheat, 4 samples of rye, and 12 samples of corn showed moniliformin in only 2 corn samples (0.06 and 0.2 micrograms/g). Moniliformin was also detected in a sample of artificially damaged (slashed) corn (0.2 micrograms/g) and selected kernels of corn that were field-inoculated with F. subglutinans and F. moniliforme (50 micrograms/g and 0.5 micrograms/g, respectively). In stability studies, moniliformin (potassium salt, 1 microgram/g) in ground corn and ground wheat heated at 50, 100, and 150 degrees C for 0.5-2 h decomposed moderately, e.g., 55% remained in corn after 0.5 h at 100 degrees C.

Formation of moniliformin by Fusarium sporotrichioides and Fusarium culmorum.

Two strains of Fusarium sporotrichioides and one strain of F. culmorum were shown to produce the mycotoxin moniliformin in rice culture. Identification was by reverse-phase liquid chromatography, thin-layer chromatography, and mass spectrometry.