Quantitative determination of T-2 toxin, HT-2 toxin, deoxynivalenol and deepoxy-deoxynivalenol in animal body fluids using LC-MS/MS detection

Abstract

A sensitive and specific method for the quantitative determination of deoxynivalenol (DON), deepoxy-deoxynivalenol (DOM-1), T-2 toxin (T-2) and HT-2 toxin (HT-2) in animal body fluids (plasma and bile) using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The extraction of plasma consisted of a deproteinization step using methanol, followed by a clean-up using an Oasis HLB solid-phase extraction column. For bile analysis, an extraction using a methanol/water mixture (70/30, v/v), followed by a liquid-liquid extraction using ethyl acetate, was performed. Chromatographic separation was achieved on a reversed-phase Nucleosil (100-5 C18 G100 × 3.0 mm) column. For the analysis of DON and DOM-1, a mixture of 0.1% acetic acid in water and methanol was used as the mobile phase. T-2 and its metabolite HT-2 were separated using 5mM ammonium acetate in a mixture of water/methanol/acetic acid. The mass spectrometer was operated in the negative or positive ESI selected reaction monitoring mode for DON and T-2 analysis, respectively. Calibration graphs (1-250 ng mL(-1)) were prepared for all matrices and correlation and goodness-of-fit coefficients were between 0.9978-1.000 and 2.96-11.77%, respectively. Limits of quantification were between 1 and 2.5 ng mL(-1) for all compounds. Limits of detection ranged from 0.01 to 0.63 ng mL(-1). The results for the within-day precision and accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of DON, DOM-1, T-2 and HT-2 in plasma and the semi-quantitative determination of the same compounds in bile from broiler chickens and pigs, respectively.