Simultaneous liquid chromatography–fluorescence analysis of type A and type B trichothecenes as fluorescent derivatives via reaction with coumarin-3-carbonyl chloride

A method for the simultaneous LC–fluorescence detection (FLD) determination of eight trichothecenes A and B by pre-column derivatization with coumarin-3-carbonyl chloride, a highly fluorescent fluorophore, has been developed. The reaction conditions (temperature, reaction time, reactant ratios) were optimized to give a reproducible quantitative conversion. All derivatives were characterized by LC–MS. The chromatographic parameters were optimized (column, eluent) to give a very good separation of three type A (diacetoxyscirpenol, T-2 toxin, HT-2 toxin) and five type B trichothecenes [deoxynivalenol (DON), nivalenol, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol]. The best conditions were obtained on a narrow-bore C18 column with a water–methanol gradient. The detection limits (S/N = 3:1) in grain samples, with an injected volume of 5 μl, were 0.2–1 ng/g for all trichothecenes. These values are more than one order of magnitude lower than those of other LC–FLD and LC–MS methods and are similar to those obtained by GC–MS. The calibration curves were linear between 100 and 2500 ng/g. The method was successfully applied to the analysis of a certified wheat reference material, after solvent extraction and clean-up on a Mycosep column, obtaining a good recovery (89% for DON) and a high accuracy (z-score value: 0.67).