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Identification of aflatoxins by quadrupole mass spectrometry/mass spectrometry.

MS/MS daughter experiments were recorded for aflatoxins B1, B2, G1, G2, M1, M2, and aflatoxicol, using 3 ionization modes. Daughters were recorded from the molecular ion (M+) using electron impact ionization (EI). Daughters from the protonated molecules (MH+) were recorded in the positive ion mode and the daughters from the molecular anion (M-) were recorded in the negative ion mode using chemical ionization (CI). These daughter spectra are all relatively simple. The EI daughters are quite similar to conventional EI spectra. The yield of (M-) is about 100 times greater than the yield of M+ in EI or MH+ in isobutane CI spectrum. Negative ion daughter spectra were used to demonstrate the feasibility of determining the presence of aflatoxin B1 in crude extracts of contaminated corn. Aflatoxin B1 could be detected at 10 ppb.

Aflatoxins↗

Effect of Piper betle L. and its extracts on the growth and aflatoxin production by Aspergillus parasiticus.

Ground powder of the leaf and fruit of Piper betle L., a tropical spice plant grown in Southeast Asia, was prepared and extracted by chloroform, ethanol and water with one solvent only or with 3 solvents in sequence. The betel powder and various extracts were added to YES broth to determine their effects on the growth and aflatoxin production by Aspergillus parasiticus. Results showed that betel leaf powder exhibited higher antimycotic activity than fruit. One half percent of ground leaf powder completely inhibited the growth and aflatoxin production by A. parasiticus. Among the solvent extracts, chloroform and ethanol extracts of betel leaf prepared from a single solvent extraction showed more antimycotic activity. The ethanol extract of betel leaf at the level of 450 micrograms/ml would eliminate A. parasiticus growth and aflatoxin production. The antimycotic activity of this ethanol extract was most pronounced at pH 4.

Aflatoxins↗

In vitro metabolism of aflatoxin Q1 by rat liver post-mitochondrial homogenates.

An investigation of the in vitro biotransformation of aflatoxin Q1 by the post-mitochondrial fraction of rat liver is reported. These studies showed a 10 to 25 percent turnover of aflatoxin Q1, with the subsequent formation of five metabolites. A major metabolite was determined to be the beta-D-glucuronide of aflatoxin Q1.

Aflatoxins↗

[Morphologic and morphometric studies of the liver of the white rat during inadequate feeding and aflatoxin poisoning].

The authors carried out morphological and morphometric studies of the liver of rats under malnutrition and chronic aflatoxin intoxication for 2 and 7 months. Malnourished animals demonstrated atrophic alterations. Animals exposed to aflatoxin B1 manifested severe dystrophic and necrobiotic alterations which did not depend on the nutrition regimen. In rats administered aflatoxin, blastomic processes were identified only at the 7th month. Whatever the diet.

Animals↗

Determination of aflatoxins in peanut butter, using two liquid chromatographic methods: collaborative study.

Two methods for determining aflatoxins in peanut butter, one using normal phase and the other reverse phase liquid chromatography (LC), were studied by 8 and 10 collaborators, respectively. Fluorescence detection was used for the determinative step in both methods. For reverse phase LC, aflatoxins B1 and G1 were converted to B2a and G2a; for normal phase LC, a silica gel-packed flow cell was placed in the irradiating light path of the detector. The samples included spiked and naturally contaminated peanut butter with total aflatoxin levels from about 5 to 20 ng/g and controls in a balanced pair design. For the normal phase LC method, recoveries of B1, B2, G1, and G2 from spiked samples averaged 79, 92, 74, and 88%, respectively; for the reverse phase method, the recoveries were 103, 104, 89, and 163%. For the normal phase LC method, pooled repeatabilities were 20, 23, 28, and 17% for B1, B2, G1, and G2, respectively; for the reverse phase method, the repeatabilities were 19, 22, 38, and 31%. For the normal phase method, pooled reproducibilities were 34, 33, 39, and 34% for B1, B2, G1, and G2, respectively; for the reverse phase method, the reproducibilities were 32, 46, 51, and 52%. Both methods show an improved limit of detection and better within-laboratory precision over current AOAC methods; however, between-laboratory precision is no better, and the reverse phase method shows evidence of interferences being measured. For these reasons and because of no benefits of present value, neither method was submitted for adoption as official first action.

Aflatoxins↗

Carcinogenic response of rainbow trout (Salmo gairdneri) to aflatoxin Q1 and synergistic effect of cyclopropenoid fatty acids.

Aflatoxin Q1 (AFQ1), the major microsomal biotransformation product of aflatoxin B1 (AFB1) formed in vitro by monkey and human liver preparations, was fed to rainbow trout (Salmo gairdneri) in a semipurified diet at levels of 20 ppb for 1 year and 100 ppb for 10 months. As a test for carcinogenicity in hatched fish, it was also used at 1 ppm in a water solution and exposed for 1 hour to fertile trout eggs. The 20-ppb dietary exposure and 1-ppm egg exposure failed to elicit a carcinogenic response; however, the 100-ppb dietary exposure produced a 10.6% incidence of hepatocellular carcinomas at the end of 1 year. Administration of 50 ppm methyl sterculate, a cyclopropenoid fatty acid, in combination with 100 ppb AFQ1 resulted in a synergistic tumor response similar to that previously noted with administration of AFB1 plus aflatoxin M1. The tumor incidence was 89.1% in the fish on the combined diet. These results indicated that AFQ1 was approximately 100 times less carcinogenic than was AFB1. This comparison of carcinogenic potencies was very similar to the comparison of the relative mutagenicities of the two compounds in the Ames bacterial mutagen assay system.

Aflatoxins↗

[Investigation of the presence of aflatoxins B1 and M1 in meat products].

Sixty-four samples of thirty-one kinds of meat products were examined by the chromatographic method with the use of a thin layer of silica gel. No aflatoxin B1 was revealed in any of the samples. Positive findings of aflatoxin M1 were obtained in three products: liver pasty, smoked rolled shoulder, and in the Spis sausage. The values found in semiquantitative evaluation were lower by 2.0 to 2.5 orders than the proposed admissible aflatoxin concentration in foods for adults in the Czech Socialist Republic (5 micrograms kg-1).

Aflatoxins↗

Rapid thin layer chromatographic determination of patulin, citrinin, and aflatoxin in apples and pears, and their juices and jams.

A method is described to determine the mycotoxins patulin, citrinin, and aflatoxins in apples and pears and their juices and jams. The mycotoxins are extracted with a mixture of acetonitrile and 4% aqueous KC1 (9 + 1). The extract is cleaned up with water and then acidified, and the toxins are recovered with chloroform and separated by thin layer chromatography. Toxin identity is confirmed with various developing solvents, spray reagents, and chemical reactions, and then quantitated by the limit of detection method. The minimum detectable concentrations of the mycotoxins are patulin, 120-130 micrograms/kg; citrinin, 30-40 micrograms/kg; aflatoxin B1 or G1, 2-2.8 micrograms/kg; aflatoxin B2 or G2, 2 micrograms/kg.

Aflatoxins↗

Survey of North Dakota sunflower seeds for aflatoxin.

In a survey of oil-type sunflower seeds, 89 samples were collected from 32 storage bins in 10 counties of North Dakota in 1979. None of these survey samples exceeded the 20 ppb Food and Drug Administration aflatoxin guideline. Four research samples, from 2 grossly spoiled storages, contained 100-1100 ppb aflatoxin B1. This research is believed to be the first to document natural aflatoxin contamination in sunflower seeds.

Aflatoxins↗

Minicolumn detection methods for aflatoxin in raw peanuts: collaborative study.

The Holaday-Velasco method and a modified Holaday method have been compared. The former method combines the speed and simplicity of the Holaday extraction and cleanup with the sensitivity of the minicolumn originally described by Velasco. The combination method has been approved by the AOAC and the AACC for determining aflatoxin in corn. The Holaday method was modified by substituting toluene for benzene in the solvent partition, and methylene chloride for chloroform in the minicolumn development to eliminate use of hazardous solvents. The neutral alumina in the Holaday minicolumn was changed from activity V to activity III to provide a more stable column. At aflatoxin levels in raw peanuts of 13-20 ng/g, the presence of aflatoxin was missed by the modified Holaday method in 4 analyses (3 laboratories) of 42 reported. There were no misses in this contamination range by the Holaday-Velasco method. There were no misses by either method with samples containing greater than 20 ng total aflatoxins/g. Analysis of uncontaminated raw peanuts by the modified Holaday method resulted in 2 false positives of 14 reports; the Holaday-Velasco method produced no false positive reports from 15 analyses of uncontaminated peanuts. The Holaday-Velasco method was adopted official first action for peanuts.

Aflatoxins↗

Reverse phase high pressure liquid chromatographic determination of aflatoxins in foods.

A method for determining aflatoxins by high pressure liquid chromatography (HPLC) with fluorescence detection after CB extraction and cleanup has been applied to various foods. Recoveries at 1--15 ppb levels from green coffee and peanut butter was 72--85 and 74--104%, respectively. Precision of the method has been tested for peanut butter. Other products to which the method has been successfully applied include tree nuts, seeds, grains, chocolate-covered peanut butter candy, and roasted, salted-in-shell peanuts. High levels of aflatoxins found in several samples of nuts by this method have been verified by the official thin layer chromatographic (TLC) method. The advantages of this HPLC method are speed, precision, sensitivity, selectivity, and immediate chemical confirmation of aflatoxins B1 and G1. None of the products analyzed required special cleanup procedures. Preparative-scale HPLC was used to isolate purified B1 for toxicity testing.

Aflatoxins↗

High pressure liquid chromatographic determination of aflatoxins in spices.

High pressure liquid chromatography with fluorescence detection is used to determine aflatoxin in 5 common spices. A 10 micrometer microparticulate silica gel column is used with a dichloromethane-cyclohexane-acetonitrile solvent system to resolve aflatoxins B1, G1, B2, and G2. The fluorescence detector contained a silica gel-packed flowcell. Samples of black, white, and red pepper, ginger, and nutmeg were extracted according to a previously published method. Recoveries from aflatoxin-free samples of white pepper, ginger, and red pepper spiked with 1-50 micrograms aflatoxin/kg ranged from 64 to 92%.

Aflatoxins↗

Dietary protein levels and aflatoxin B metabolism in rainbow trout (Salmo gairdneri).

In an attempt to understand dietary protein effects upon aflatoxin B1-induced liver cancer in rainbow trout, the activities of several suspected aflatoxin B1 metabolizing enzyme systems were studied relative to protein intake. Fish fed diets containing 32 percent, 52 percent and 62 percent fish protein concentrate (FPC) were examined for hepatic cytochrome P-450 content and in vitro cytochrome c reductase, glutathione-S-epoxide transferase (GTr), epoxide hydrase (EH) and aldrin epoxidase (AE) activity. In addition, aflatoxin B1 conversion to aflatoxicol (AFL) was examined. A direct correlation was observed between increased FPC intake and cytochrome P-450 content and AFL production, with increases of 14 percent and 41 percent respectively. With increased FPC intake, decreases in EH (15 percent), GRr (20 percent), cytochrome c reductase (13 percent) and AE (50 percent) activities were noted. These findings are discussed in relation to AFB feeding trials reported earlier.

Aflatoxins↗

Genetic resistance of chickens to aflatoxin assessed with organ-culture techniques.

A chick tracheal-organ-culture model system was used to evaluate the sensitivity of various strains of chickens to aflatoxin B1. Tissues from different genetic stocks showed highly significant differences in in vitro sensitivity to aflatoxin. Tracheal tissues from brown-egg layers were most sensitive, meat-type birds intermediate, and white-egg layers most resistant. Within-category strains and sex differed significantly in sensitivity to damage by aflatoxin, with male chicks more resistant than female chicks.

Aflatoxins↗

Activation and inhibition of benzo(a)pyrene and aflatoxin B1 metabolism in human liver microsomes by naturally occurring flavonoids.

The effects of several naturally occurring and synthetic flavonoids on the metabolism of benzo(a)pyrene and aflatoxin B1 were evaluated. Addition of apigenin, chrysin, fisetin, flavonone, galangin, hesperitin, kaempferol, morin, myricetin, haringenin, or quercetin to human liver microsomes inhibited the hydroxylation of benzo(a)pyrene. In contrast to these results, the addition of flavone, nobiletin, tangeretin, or 7,8-benzoflavone to human liver microsomes caused a many-fold stimulation in the hydroxylation of benzo(a)pyrene, the metabolism of aflatoxin B1 to 2,3-dihydro-2,3-dihydroxyaflatoxin B1, and the metabolic activation of aflatoxin B1 to mutagenic products. Quercetin, morin, and kaempferol inhibited cytochrome c (P-450) reductase in human liver microsomes whereas flavone and 7,8-benzoflavone had no effect. These results suggest that the inhibitory effects of quercetin, morin, and kaempferol on monooxygenase activity may be caused at least in part by an inhibition in the reduction of cytochrome P-450. An examination of the structural features required for the inhibition and stimulation of benzo(a)pyrene hydroxylation indicated that all of the 12 flavonoid inhibitors that were studied possessed hydroxyl groups whereas the flavonoid activators were less polar molecules that lacked hydroxyl groups.

Aflatoxins↗

Comparison of three methods for determining aflatoxins in sunflower seed meals.

Methods developed for the determination of aflatoxins in peanut products, in cottonseed products, and in ginger were compared for their applicability to sunflower seed meal. The peanut products method was inapplicable. Separation from interferences adequate to obtain a limit of determination for aflatoxin B1 around 1 ng/g and recovery of total added aflatoxins of about 97% could be achieved with either the cottonseed or ginger methods. The cleanest extract and the most rapid operation were obtained with the ginger method.

Aflatoxins↗

Binding characteristics of bovine serum albumin-aflatoxin B1 to polystyrene microtiter plates: importance of hapten to carrier protein molar ratio.

Binding characteristics of bovine serum albumin-aflatoxin B1 conjugates with high (1:54), medium (2:25) and low (1:9) hapten to carrier molar ratios, to the polystyrene microtiter plates are influenced by the stoichiometry of the hapten (Aflatoxin B1) to the carrier protein (bovine serum albumin). Conjugates with optimal hapten to carrier molar ratios (1:25) showed a better binding capacity to the polystyrene microtiter plate as compared to the conjugates with the high molar ratios in a non-competitive ELISA for aflatoxin B1. Denaturation of the conjugate with molar ratio of 1:54 in order to enhance its binding capacity, however, did not result in any significant improvement.

Aflatoxin B1↗

The short-term effect of low concentrations of dietary aflatoxin and T-2 toxin on mallard ducklings.

Two 9-day trials to study effects of dietary mycotoxins were conducted using 6-week-old mallards. Each trial involved three groups of six ducks each, three males and three females per group. One group was fed no myocotoxin (control), one was fed aflatoxin, and the third was fed T-2 toxin. Dietary aflatoxin was 12 ppb in trial 1 and 33 ppb in trial 2; dietary T-2 toxin was 2 ppm in both trials. There were no gross or microscopic lesions in controls or in ducks fed aflatoxin, nor did these groups differ significantly in final body weight or in weights of spleen or bursa of Fabricius. Ducks exposed to T-2 toxin had erosions and/or ulcerations in the oral cavity and esophagus; further, these ducks exhibited a decrease in weights of body, thymus, spleen, and bursa of Fabricius. The only microscopic change seen in lymphoid organs of ducks exposed to T-2 toxin was a moderate decrease of thymic cortical lymphocytes.

Aflatoxins↗