Preparative Separation and Purification of Trichothecene Mycotoxins from the Marine Fungus Fusarium sp. LS68 by High-Speed Countercurrent Chromatography in Stepwise Elution Mode.

The contamination of foods and animal feeds with trichothecene mycotoxins is a growing concern for human and animal health. As such, large quantities of pure trichothecene mycotoxins are necessary for food safety monitoring and toxicological research. A new and effective method for the purification of trichothecene mycotoxins from a marine fungus, Fusarium sp. LS68, is described herein. Preparative high-speed countercurrent chromatography (HSCCC) was utilized for the scalable isolation and purification of four trichothecene mycotoxins for the first time in stepwise elution mode, with a biphasic solvent system composed of hexanes-EtOAc-CH₃OH-H₂O (6:4:5:5, v / v / v / v ) and (8.5:1.5:5:5, v / v / v / v ). This preparative HSCCC separation was performed on 200 mg of crude sample to yield four trichothecene mycotoxins, roridin E ( 1 ), roridin E acetate ( 2 ), verrucarin L acetate ( 3 ), and verrucarin J ( 4 ) in a single run, with each of >98% purity. These compounds were identified by MS, ¹H NMR, 13 C NMR, and polarimetry. The results demonstrate an efficient HSCCC method for the separation of trichothecene mycotoxins, which can be utilized to produce pure commercial and research standards.