HPLC assay of zearalenone and reduced metabolites in S9 fractions of duck liver

Abstract

HPLC analysis of zearalenone (ZEA), zearalenols (-ZOL and ß-ZOL) and zearalanols (-ZAL and ß-ZAL) was developed, in order to obtain a sensitive and reproducible method to quantify ZEA and its reduced metabolites in subcellular fractions of animal livers (S9 samples). Optimal in vitro metabolism was observed by incubating 5 mg S9 proteins with 0.016 μmol. ZEA. Acetonitrile and diethylether/chloroform mixture were compared for extraction, as well as different mobile phases and two detection modes in HPLC analysis. Extracted samples were eluted with water/acetonitrile (55:45, v/v) at a flow-rate of 1.0 ml/min-1, resulting in well separated peaks between ZEA and the metabolites. The limits of detection ranged from 0.5 to 2 ng/mg S9 proteins using UV, and from 0.04 to 4 ng/mg S9 proteins, using fluorescence detection. Fluorescence showed a ten-fold higher sensitivity than UV detection for ZEA and -ZOL. Repeatability (10 assays) was 2.7% to 6.99% for zearalenols. Day-by-day coefficients of variation for zearalenone and zeranols with UV detection were 3.3 to 8.5 %, and 2.5 to 4.3 %, respectively. This analysis applied to S9 samples from ducks after 30 min of ZEA incubation allowed to demonstrate that -ZOL is the main reduced metabolite in the duck. The present method is particularly adapted for studying in vitro metabolism of ZEA and inter-species variations.