E.W. Sydenham

Quantitative Determination of Fumonisins B1 and B2 by High-Performance Liquid Chromatography with Fluorescence Detection

Abstract A high-performance liquid chromatographic method has been developed for the quantitative determination of the recently described mycotoxins, fumonisins B1 (FB1) and B2 (FB2) utilizing pre-column derivatization with o-phthaldialdehyde, isocratic elution, and fluorescence detection. Prior to analysis, sample extracts were purified on strong anion exchange cartridges. The method has been applied to the analysis of naturally contaminated corn and mixed horse feed samples as well as fungal culture material, for the presence of the mycotoxins. Detection limits are approximately 50 ng g−1 for FB1 and 100 ng g−1 for FB2. The method proved to be highly reproducible and recoveries of the toxins from the purification steps were found to be 99.5% and 85.9% for FB1 and FB2, respectively.

Fate of a single dose of the 14C-labelled mycotoxin, fumonisin B1, in rats

The fate of the mycotoxin, fumonisin B1, (FB1) dosed to rats by i.p. injection and by gavage was traced using 14C-labelled FB1. Twenty-four hours after i.p. injection, 66% of the radioactivity was recovered in faeces, 32% in urine, 1% in liver and trace amounts (less than 1%) in kidney and red blood cells. When dosed by gavage, all (101%) radioactivity was recovered in faeces and trace amounts were found in urine, liver, kidney and red blood cells. The bulk of the radioactivity recovered was unmetabolized FB1.

Determination of the mycotoxin fumonisin B1 and identification of its partially hydrolysed metabolites in the faeces of non-human primates

A method has been developed for the determination of fumonisin B1 (FB1) in the faeces of non-human primates (vervet monkeys). The animals were dosed with 14C-labelled FB1, and the radioactive compounds in faeces were recovered by repeated extractions with 0.1 M ethylenediaminetetraacetic acid. The extracts were cleaned-up on a reversed-phase (C18) solid-phase extraction cartridge, and FB1 was determined by o-phthaldialdehyde derivatization and reversed-phase HPLC. The analytical method for the determination of FB1 in the faecal extracts was reproducible [2.6% relative standard deviation (RSD)] and accurate (recovery from spiked blank extracts of 93 +/- 2.9% RSD). Confirmation of the identification of FB1 in faeces was achieved using HPLC and thin-layer chromatography, which showed that the radioactivity extracted corresponded mainly to FB1 and a new metabolite with chromatographic properties similar to those of the mycotoxin. The new metabolite was identified by mass spectrometry and nuclear magnetic resonance spectroscopy to be an equilibrium mixture of the two structural isomers of partially hydrolysed FB1, which are formed by hydrolysis of one of the ester groups of the mycotoxin.

Distribution and excretion of a single dose of the mycotoxin fumonisin B1 in a non-human primate

Fumonisin B1 (FB1), a toxic and carcinogenic secondary metabolite of the fungus Fusarium moniliforme Sheldon, was administered either by i.v. injection or by gavage to vervet monkeys (Cercopithecus aethiops). FB1 dosed by i.v. injection to two female vervet monkeys was rapidly eliminated from plasma with a mean half-life during the elimination phase of 40 min. Analysis of urine and faeces over a 5 day period after dosing gave an average 47% recovery of the dose as FB1 and its hydrolysed analogues. Two female vervet monkeys were given a single gavage dose of 14C-labelled FB1. During the subsequent 3 day period, faecal excretion of radioactivity accounted for an average of 61% of the administered dose and urinary excretion 1.2%. Residual radioactivity was recovered in low levels from skeletal muscle (1%), liver (0.4%), brain (0.2%), kidney, heart, plasma, red blood cells and bile (each 0.1%), while the contents of the intestines accounted for a further 12% of the radioactive dose. In total, 76% of the administered radioactivity was recovered. Analysis of the faeces, intestinal contents and urine indicated that over 90% of the radioactivity in these samples was due to FB1 and its hydrolysis products.

Initial studies on the toxicokinetics of fumonisin B1 in rats

Fumonisin B1 (FB1), the major compound in the fumonisin group of secondary metabolites of Fusarium moniliforme Sheldon, is associated with some human and animal diseases. After intraperitoneal dosing to rats (7.5 mg/kg), FB1 was rapidly absorbed and reached a maximum concentration in plasma within 20 min after injection. Thereafter, it underwent rapid removal from plasma, displaying a mono-exponential elimination phase that fitted a one-compartment model with a half-life of 18 min. Collection of 24- and 48-hr urine samples indicated that only 16% of the applied dose was eliminated unmetabolized in urine, all within the first 24-hr period following dosing. In contrast to this, a similar dose of FB1 given by gavage resulted in the recovery of only 0.4% of the FB1 in urine.

Production of mycotoxins by selected Fusarium graminearum and F. crookwellense isolates

Corn cultures (five isolates each of Fusarium graminearum Group 1 from wheat crowns, Group 2 from scabby wheat grains and from ear rot of corn and five isolates of F. crookwellense) were screened for their ability to produce deoxynivalenol (DON), nivalenol (NIV), fusarenon-x (FUS-X) and zearalenone (ZEA). Nine of the ten F. graminearum isolates from wheat produced DON (5-165 micrograms g-1) but none produced either NIV or FUS-X. Conversely, 3/5 and 2/5 of the F. graminearum isolates from corn produced NIV (5-40 micrograms g-1) and FUS-X (5-7 micrograms g-1), respectively, while none produced DON. All but one of the F. graminearum isolates produced ZEA (2-1160 micrograms g-1). None of the F. crookwellense isolates produced DON, but 5/5 and 4/5 produced NIV (6-170 micrograms g-1) and FUS-X (3-90 micrograms g-1), respectively, and all produced ZEA (605-1030 micrograms g-1). The results confirmed previous findings on the presence of two distinct F. graminearum chemotypes.

Reversed-phase high-performance liquid chromatography of tenuazonic acid and related tetramic acids.

A reversed-phase high-performance liquid chromatographic system for the determination of the fungal toxin, tenuazonic acid, (5S,8S)-3-acetyl-5-sec.-butyltetramic acid, is described. The system utilizes a column packed with deactivated end-capped C18 silica with a high carbon load to overcome the problem of poor chromatographic performance of this beta-diketone on reversed-phase liquid chromatography which previously necessitated the use of anion-exchange, ligand-exchange or ion-pairing methods. The reversed-phase system allows the separation of tenuazonic acid from its (5R,8S)-diastereomer, allo-tenuazonic acid and was applied to the detection of tenuazonic acid in cultures of Alternaria alternata and Phoma sorghina. By means of diode-array ultraviolet detection, (5S)-3-acetyl-5-isopropyltetramic acid was observed in extracts of culture material. This metabolite was purified using the analytical reversed-phase system and was identified by 1H and 13C nuclear magnetic resonance spectroscopy.

Determination of fumonisin B1 in plasma and urine by high-performance liquid chromatography

Fumonisin B1 (FB1), the major compound of the newly described fumonisin mycotoxins, has been shown to be the causative agent of the animal diseases leukoencephalomalacia in horses and pulmonary oedema in pigs. Whereas previous analytical methods have dealt with the determination of FB1 in feed and foodstuffs, this report for the first time details methods for FB1 determination in the physiological fluids, plasma and urine. The methods involve solid-phase anion-exchange clean-up, precolumn derivatisation with o-phthaldialdehyde and reversed-phase high-performance liquid chromatography with fluorescence detection. These methods were shown to be sensitive (detection limit around 50 ng ml-1), reproducible (relative standard deviation on six replicates less than 5%) and accurate (recoveries on spiked blank samples above 85%).

A Mycological Evaluation and in Vivo Toxicity Evaluation of Feed from 41 Farms with Equine Leukoencephalomalacia

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Toxicity and moniliformin production by four recently described species of Fusarium and two uncertain taxa

Four recently described species, Fusarium nygamai, F. dlamini, F. beomiforme and F. napiforme and two uncertain taxa, F. nygamai from millet in Africa and Fusarium species from rice with Bakanae disease, were tested for toxicity and moniliformin production. Cultures grown on autoclaved corn were fed to groups of four one-day-old ducklings for 14 days. Isolates that caused the death of 3 or 4 out of 4 ducklings were considered to be toxic and analyzed for moniliformin. All 15 isolates of F. dlamini tested were nontoxic. The other taxa contained some isolates that were toxic to ducklings and produced moniliformin in corn cultures. This is the first report of moniliformin production by F. beomiforme (200–890 μg/g), and F. napiforme (16–388 μg/g), and by F. nygamai not obtained from millet in Africa (15–874 μg/g). The highest production of moniliformin was obtained from the 19 isolates of F. nygamai from millet in Africa (4300–18200μg/g) and the 15 isolates from rice with Bakanae disease (2300–19300 μg/g). The taxonomic position of these two uncertain taxa should be re-evaluated.