A Rapid and Sensitive Procedure for the Quantitative Detection of AL-toxin by Fluorescence Derivatization and Separation by High Performance Liquid Chromatography.

An improved high performance liquid chromatographic (HPLC) method was developed for the detection and quantitation of host-specific AL-toxin produced by Alternaria alternata tomato pathotype. Precolumn derivatization of the toxin with o-phthalaldehyde (OPA) plus mercaptoethanol or 4-fluoro-7-nitrobenzofurazan (NBD-F) yielded highly fluorescent products showing well-resolved peaks on reversed phase HPLC. The minimum amounts of AL-toxins detectable by this method were approximately 1ng. This method seems to offer advantages over conventional techniques, because prechromatographic derivatization of the toxin with OPA or NBD-F is rapid and easy, and fluorescent derivatives permit excellent detection sensitivity. Toxin production in culture filtrates and spore-germinated fluids of the pathogen was quantitatively analyzed using this procedure.