Improvement of detection sensitivity of T-2 and HT-2 toxins using different fluorescent labeling reagents by high-performance liquid chromatography

Abstract

T-2 and HT-2 toxins are Fusarium mycotoxins that can occur in cereals and cereal-based products. Three fluorescent labeling reagents, i.e. 1-naphthoyl chloride (1-NC), 2-naphthoyl chloride (2-NC) and pyrene-1-carbonyl cyanide (PCC), were used for the determination of T-2 and HT-2 toxins by high-performance liquid chromatography (HPLC) with fluorescence detection (FD). Pre-column derivatization of T-2 and HT-2 toxins was carried out under mild conditions (50 °C, 10 min) in toluene with 4-dimethylaminopyridine (DMAP) as catalyst. All fluorescent derivatives were identified and characterized by HPLC-tandem mass spectrometry (HPLC-MS/MS). Optimal stoichiometric ratios (toxin:derivatizing reagent:catalyst), linear range and repeatability of the reaction, stability and sensitivity of the derivatives were determined. A wide linear range (10–1000 ng of either derivatized T-2 or HT-2 toxin), good stability (up to 2 weeks at −20 °C or 5 days at room temperature) of the fluorescent derivatives and good repeatability of the reaction (RSD ≤ 8%) were observed. Detection limits (based on a signal-to-noise ratio of 3:1) were 10.0, 6.3 and 2.0 ng for derivatized T-2 toxin and 6.3, 2.3 and 2.8 ng for derivatized HT-2 toxin with 1-NC, 2-NC and PCC, respectively. In terms of sensitivity and repeatability, PCC and 2-NC reagents showed better performance than 1-anthroylnitrile (1-AN), a previously reported labeling reagent for T-2- and HT-2 toxins. Preliminary studies also showed the applicability of PCC and 2-NC as fluorescent labeling reagents for the simultaneous determination of T-2 and HT-2 toxins in cereal grains by HPLC/FD following immunoaffinity column clean-up.