Determination of type A trichothecenes by high-performance liquid chromatography with coumarin-3-carbonyl chloride derivatisation and fluorescence detection

A method for the analysis of type A trichothecenes T-2 toxin, HT-2 toxin, n eosolaniol and diacetoxyscirpenol by high-performance liquid chromatography with fluorescence detection using coumarin-3-carbonyl chloride has been de veloped. Different parameters concerning the analytical procedure such as s tability of both the reagent and derivatised analytes, time and temperature of the derivatisation reaction, were studied and optimised. Three differen t clean-up procedures (solid-phase extraction with silica gel or C-18 cartr idges, and Liquid-liquid partition between toluene and dihydrogen phosphate buffer) were tested in order to remove the excess reagent peaks. The last procedure gave the best results when the buffer pH was 3-5.5, and is theref ore recommended. Separations were performed on a stainless steel LiChrosphe r 100 C-18 reversed-phase column with pre-column of the same phase. The mob ile phase was acetonitrile/water (65:35, v/v) containing 0.75% acetic acid at a flow-rate of 1.0 ml/min. The proposed method provides good separation between the four trichothecenes and good reproducibility (RSD of calibratio n standards <5%), The Limits of detection of the studied trichothecenes at a signal-to-noise ratio of 3:1, with an injection volume of 20 mu l were 10 ng/g sample for T-2 toxin and about 15 ng/g sample for the remaining mycot oxins. The calibration curve was linear between 10 and 2000 ng for the four trichothecenes assayed. The method was applied to the analysis of these my cotoxins in fungal cultures (corn and rice) of Fusarium sporotrichioides, a nd is also perfectly suitable for the quantification of type A trichothecen es in contaminated cereals. (C) 2000 Elsevier Science B.V. All rights reser ved.