Tracing the metabolism of HT-2 toxin and T-2 toxin in barley by isotope-assisted untargeted screening and quantitative LC-HRMS analysis.

Jacqueline Meng-Reiterer,Alexis V Nathanail,Gerhard Adam,Herbert Michlmayr,Susan P Mccormick,Justyna Rechthaler,Philipp Fruhmann,Marc Lemmens,Elisabeth Varga,Elisabeth Varga,Rainer Schuhmacher,Christoph Bueschl,Franz Berthiller,Franz Berthiller,Alexandra Malachová,Alexandra Malachová

doi:10.1007/s00216-015-8975-9

Abstract

An extensive study of the metabolism of the type A trichothecene mycotoxins HT-2 toxin and T-2 toxin in barley using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) is reported. A recently developed untargeted approach based on stable isotopic labelling, LC-Orbitrap-MS analysis with fast polarity switching and data processing by MetExtract software was combined with targeted LC-Q-TOF-MS(/MS) analysis for metabolite structure elucidation and quantification. In total, 9 HT-2 toxin and 13 T-2 toxin metabolites plus tentative isomers were detected, which were successfully annotated by calculation of elemental formulas and further LC-HRMS/MS measurements as well as partly identified with authentic standards. As a result, glucosylated forms of the toxins, malonylglucosides, and acetyl and feruloyl conjugates were elucidated. Additionally, time courses of metabolite formation and mass balances were established. For absolute quantification of those compounds for which standards were available, the method was validated by determining apparent recovery, signal suppression, or enhancement and extraction recovery. Most importantly, T-2 toxin was rapidly metabolised to HT-2 toxin and for both parent toxins HT-2 toxin-3-O-β-glucoside was identified (confirmed by authentic standard) as the main metabolite, which reached its maximum already 1 day after toxin treatment.Graphical Isotope-assisted untargeted screening of HT-2 toxin and T-2 toxin metabolites in barleyElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-8975-9) contains supplementary material, which is available to authorized users.

Highlights

HT-2 toxin (HT2) and T-2 toxin (T2) are secondary metabolites of fungi belonging to the genus Fusarium and are classified as type A trichothecene mycotoxins
The qualitative screening of HT2 and T2 metabolites in barley was generally based on treatment of barley ears with a mixture of non-labelled and uniformly 13C-labelled toxin, extraction, measurement of sample extracts with LC-Orbitrap-MS in fast polarity switching mode and data processing by MetExtract
Measurements in fast polarity switching mode showed that HT2 biotransformation products were found in both applied polarities, and different adducts were formed which facilitated the annotation of the molecular identity of the ion species and increased the probability of MetExtract detection of metabolites with low abundance

Summary

Introduction

HT-2 toxin (HT2) and T-2 toxin (T2) are secondary metabolites of fungi belonging to the genus Fusarium and are classified as type A trichothecene mycotoxins. Oats, wheat and barley, are especially affected by type A trichothecene-producing fungi and are prone to contamination with HT2 and T2 [1, 2]. Plants employ various detoxification mechanisms to cope with the adverse effects of mycotoxins. Understanding the plant metabolism of mycotoxins and the resulting metabolic derivatives is becoming increasingly important for risk assessment. Mirocha et al [7] reported the occurrence of HT2, T-2-tetraol, 3′-hydroxy-HT-2 and 3′-hydroxy-T-2 formed in T2-treated Baccharis species. Whilst the work presented here details the technical aspects of metabolite detection and characterisation, the study mentioned above focuses on the biological interpretation of HT2 and T2 metabolism in wheat and complements the presented study

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