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

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. 1. 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 [1–3]. Due to its pronounced estrogenic activity, the exposure to ZEN has been associated with endocrine disruptive effects in domestic animals, especially in pigs, and possibly in humans [1, 4]. As is the case with virtually all other mycotoxins, ZEN is not the only congener produced by the fungus, and several other fungal resorcylic acid lactones have previously been identified, in particular the α- and β-stereoisomers of zearalenol (ZEL, Figure 1) and of 5-hydroxy-ZEN and 10-hydroxy-ZEN [5]. More recently, ZEN-11,12-oxide, ZEN-11,12-dihydrodiol and cyclization products of the latter have been reported as fungal metabolites [5]. Figure 1: Chemical structures of ZEN and ZEL. Arrows indicate positions of hydroxylation in fungal (f) and mammalian (m) metabolites. Nomenclature according to [ 6]. In addition to fungal metabolites of ZEN, numerous monohydroxylation products of ZEN and ZEL have recently been disclosed as mammalian metabolites in vitro [7–9]. Depending on the species, either the aromatic ring or the aliphatic macrocycle were the preferred site for hydroxylation (Figure 1). Taken together, 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. Although the