|Lippolis, Vincenzo - INST. SCIENCES FOOD PROD.|
|Pascale, Michelangelo - INST. SCIENCES FOOD PROD.|
|Visconti, Angelo - INST. SCIENCES FOOD PROD.|
Submitted to: Talanta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 2, 2007
Publication Date: January 12, 2008
Citation: Lippolis, V., Pascale, M., Maragos, C.M., Visconti, A. 2008. Improvement of detection sensitivity of T-2 and HT-2 toxins using different fluorescent labeling reagents by high-performance liquid chromatography. Talanta. 74(5):1476-1483. Interpretive Summary: T-2 toxin is a mycotoxin produced by several species of common fungi capable of infesting human food and animal feeds. T-2 toxin and its metabolite HT-2 are acutely toxic to animals. Sensitive detection of T-2 and HT-2 toxins is somewhat limited because these compounds do not fluoresce and do not have a strong ultraviolet or visible absorption band. This report describes the development of sensitive methods to detect both T-2 toxin and HT-2 toxins by labeling them with one of several novel fluorescent tags. The tagged T-2 and HT-2 toxins are very stable and the fluorescence response of the tagged toxins is an improvement over previous labeling materials, resulting in more sensitive assays for these toxins.
Technical Abstract: T-2 and HT-2 toxins are Fusarium mycotoxins that can occur in cereals and cereal-based products. Three novel 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, and 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 less than or equal to 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 the best performance. 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.