Combination of LC-MS2 and GC-MS as a Tool to Differentiate Oxidative Metabolites of Zearalenone with Different Chemical Structures

Andreas A Hildebrand,Manfred Metzler,Erika Pfeiffer,Georg Damm

doi:10.1155/2012/472031

Abstract

Recent studies on the mammalian and fungal metabolism of the mycotoxin zearalenone (ZEN) have disclosed the formation of six regioisomers of monohydroxy-ZEN and its reductive metabolite zearalenol (ZEL). Hydroxylation occurs at the aromatic ring or at one of four positions of the aliphatic macrocycle. In addition, an aliphatic ZEN epoxide, its hydrolysis product, and other products were identified in fungal cultures. In this paper, we report the product ion spectra of the [M-H]− ions of 22 oxidative metabolites of ZEN and ZEL, obtained by LC-MS2 analysis using a linear ion trap mass spectrometer with negative electrospray ionization. The MS2 spectra exhibit qualitative and quantitative differences which allow a clear distinction of most metabolites. Moreover, GC-MS analysis of the trimethylsilylated metabolites yields electron impact mass spectra with numerous fragment ions which can be used as fingerprint to confirm the chemical structure derived by LC-MS2 analysis.

Highlights

Zearalenone (ZEN) is a mycotoxin with the chemical structure of a β-resorcylic acid lactone (Figure 1), which is produced by Fusarium species and frequently found as a contaminant of food and feed [1,2,3]
Either the aromatic ring or the aliphatic macrocycle were the preferred site for hydroxylation (Figure 1)
Hydroxyl groups located at C-5, C-6, C-8, or C-9 are lost during this fragmentation, whereas hydroxyl groups at C-10, C-13, or C-15 are retained in the fragment, shifting its mass to m/z 191 (Figure 2)

Summary

Introduction

Zearalenone (ZEN) is a mycotoxin with the chemical structure of a β-resorcylic acid lactone (Figure 1), which is produced by Fusarium species and frequently found as a contaminant of food and feed [1,2,3]. ZEN-11,12-oxide, ZEN-11,12-dihydrodiol and cyclization products of the latter have been reported as fungal metabolites [5]. In addition to fungal metabolites of ZEN, numerous monohydroxylation products of ZEN and ZEL have recently been disclosed as mammalian metabolites in vitro [7,8,9]. Fourteen oxidative ZEN and ZEL metabolites are presently known as regioisomers, that is, differing in the position of the hydroxyl group. The total number of oxidative metabolites is even higher, as hydroxylation at each aliphatic position gives rise to two stereoisomers. In the course of our past LC-MS2 studies using a linear trap mass spectrometer [5, 8, 9], we observed that fragmentation of the [M-H]− ions obtained by electrospray ionization in the negative mode gives rise to product ion spectra which differ significantly between different regioisomers. We propose to use a combination of LC-MS2 and GC-MS for the identification of oxidative ZEN metabolites in the analysis of food samples and body fluids

Mass Spectrometry

Results

Discussion