Novel methods for determination of the trichothecene mycotoxin deoxynivalenol

Abstract

A rapid method for analysis of deoxynivalenol (DON) was developed using high pressure liquid chromatography with reductive electrochemical detection (LCECD). Deoxynivalenol produced by Fusarium roseum growing on solid cornmeal and rice substrates and from naturally contaminated wheat was extracted and quantitated. DON levels in wheat were verified by gas chromatography and structurally confirmed by mass spectrometry. DON was optimally resolved on LC employing a radially compressed octadecylsilane column and a mobile phase of deoxygenated methanol-40 mM borate buffer (30:70) at a flow rate of 1.0 ml/min. Under these conditions DON exhibited an average retention time of 3-6 min. A 12-fold increase in sensitivity and more selectivity was observed over classical UV absorption at 225 nm employing reductive on-line electrochemical detection (i.e glassy carbon electrode and an applied voltage of -1.4 V). A detection limit for DON of 0.25 ppb was achieved under these conditions. The determination of DON in crude grain extracts was hindered by extractable interfering substances; whereas, electrochemical detection was more functional group selective (i.e. reduction of the carbonyl moiety). LCECD permitted a direct quantitation of DON from crude grain extracts and may facilitate the determination of this agent and associated metabolites in biological samples. Two deoxynivalenol producing isolates of Fusarium roseum (ATCC #28114, NCPRL-A) were incubated in stationary liquid culture in the presence of [1-^14C] sodium acetate to biosynthetically produce a radiolabeled toxin suitable for use in tissue distribution studies. Specific activities of 139 uCi/mmole (NCPRL-A on corn media), 67.5 uCi/mmole (NCPRL-A on rice media) and 21.5 uCi/mmole (ATCC #28114 on rice media) were obtained in this study and represent the first reported incorporation of ^14C into deoxynivalenol...