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A fast and sensitive gas chromatographic/negative ion chemical ionization mass spectrometric method for the simultaneous detection and quantification of several simple trichothecene mycotoxins and related molecules with good precision has been developed. The method consists of the conversion of the polar trichothecenes into their volatile and stable heptafluorobutyryl esters, followed by gas chromatographic/mass spectrometric analysis of the derivatives. The derivatives were ionized under chemical ionization conditions and the negative ions were monitored. The samples were screened for the presence of trichothecenes by monitoring one characteristic ion by selected ion monitoring for each analyte. The detected compounds were confirmed by monitoring 5-6 characteristic ions by selected ion monitoring whenever the amount of analyte present was insufficient to obtain a full scan negative ion chemical ionization mass spectrum. Two semi-synthetic trichothecenes, 4-deoxyverrucarol and 16-hydroxyverrucarol, were investigated and found to be adequate internal standards both for detection and quantification of these toxic compounds. Femtogram and low picogram (1-5 pg) quantities of these compounds could be detected and confirmed, respectively, by this procedure. A short clean-up procedure using Sep-Pak (silica gel) cartridges was developed and found to be applicable for the analysis of some real samples. Several spiked and real samples were analysed by this method, with excellent sensitivity and precision.

1. The effects of safrole and isosafrole pretreatment on both N- and ring-hydroxylation of 2-acetamidofluorene were studied in male rats and hamsters. 2. Isosafrole (100mg/day per kg body wt.) pretreatment of rats for 3 days did not have any effect on urinary excretion of hydroxy metabolites of 2-acetamidofluorene. However, similar pretreatment with safrole produced increased urinary excretion of N-, 3- and 5-hydroxy derivatives. 3. Similar treatment with these two chemicals for 3 days increased ring-hydroxylation activity by rat liver microsomal material. Increases in N-hydroxylation were much less than those in ring-hydroxylation. Isosafrole was twice as effective as safrole. 4. Increases in hydroxylating activity due to safrole or isosafrole treatment were inhibited by simultaneous administration of ethionine. Similarly, ethionine inhibition was almost completely reversed by the simultaneous administration of methionine. 5. Safrole or isosafrole (0.1mm and 1mm) inhibited 7-hydroxylation activity by liver microsomal material from control rats. At 1mm these two chemicals inhibited both 5- and 7-hydroxylation activity by liver microsomal material from 3-methylcholanthrene-pretreated rats. 3-Hydroxylation activity was not inhibited by 1mm concentrations of these two chemicals. 6. A single injection of safrole (50100 or 200mg/kg body wt.) 24h before assay had no appreciable effect on either N- or ring-hydroxylation activity by hamster liver microsomal material. However, isosafrole (200mg/kg body wt.) treatment inhibited N-, 3- and 5-hydroxylation activities by hamster liver microsomal material; it had no effect on 7-hydroxylation activity.

1. Reaction of 2-(N-acetoxy)-acetamidofluorene with orthophosphate buffer at pH7 yielded a large quantity of water-soluble fluorene derivatives, which showed absorption peaks at 303, 290 and 280nm. Tris buffer under similar conditions gave negligible reaction. 2. Hydrolysis of polar material with acid or alkaline phosphatases liberated equimolar amounts of inorganic phosphate and an ether-extractable fluorene derivative. On the basis of its u.v. spectrum, R(F) values after paper chromatography, solubility in alkali and colour with spray reagents, the derivative was characterized tentatively as 2-acetamido-5-hydroxyfluorene. 3. Polar material also contained a reactive fluorene derivative which gave characteristic reaction products with methionine and guanosine. The reactive derivative was characterized as a phosphate ester of 2-(N-hydroxy)-acetamidofluorene. 4. It is suggested that such reactive phosphate esters may also be some of the ultimate carcinogenic metabolites of carcinogenic aromatic hydroxamic acids.

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