Abstract
An untargeted screening strategy for the detection of biotransformation products of xenobiotics using stable isotopic labelling (SIL) and liquid chromatography–high resolution mass spectrometry (LC-HRMS) is reported. The organism of interest is treated with a mixture of labelled and non-labelled precursor and samples are analysed by LC-HRMS. Raw data are processed with the recently developed MetExtract software for the automated extraction of corresponding peak pairs. The SIL-assisted approach is exemplified by the metabolisation of the Fusarium mycotoxin deoxynivalenol (DON) in planta. Flowering ears were inoculated with 100 μg of a 1 + 1 (v/v) mixture of non-labelled and fully labelled DON. Subsequent sample preparation, LC-HRMS measurements and data processing revealed a total of 57 corresponding peak pairs, which originated from ten metabolites. Besides the known DON and DON-3-glucoside, which were confirmed by measurement of authentic standards, eight further DON-biotransformation products were found by the untargeted screening approach. Based on a mass deviation of less than ±5 ppm and MS/MS measurements, one of these products was annotated as DON-glutathione (GSH) conjugate, which is described here for the first time for wheat. Our data further suggest that two DON-GSH-related metabolites, the processing products DON-S-cysteine and DON-S-cysteinyl-glycine and five unknown DON conjugates were formed in planta. Future MS/MS measurements shall reveal the molecular structures of the detected conjugates in more detail.
Highlights
Xenobiotics are frequently metabolised and subsequently conjugated to more polar derivatives as a part of detoxification strategies of organisms, including animals and plants
Stable isotopic labelling (SIL)-assisted metabolomics techniques [5,6,7,8,9] offer a wide range of new applications in the field of untargeted approaches
A major challenge is the detection of the labelled biotransformation products within highly complex liquid chromatography–high resolution mass spectrometry (LC-HRMS) chromatograms
Summary
Xenobiotics are frequently metabolised and subsequently conjugated to more polar derivatives as a part of detoxification strategies of organisms, including animals and plants. SIL-assisted approaches provide additional information on the number of carbon atoms of the detected metabolite ions [5]. Both stable isotopic labelled endogenous [10] as well as xenobiotic [3, 11] precursors have been used to study in vivo metabolisation in biological samples. In this context, a major challenge is the detection of the labelled biotransformation products within highly complex liquid chromatography–high resolution mass spectrometry (LC-HRMS) chromatograms. The recently developed software algorithm MetExtract [12] is capable of the automated global detection of metabolite-derived LC-HRMS signals originating from non-labelled and stable isotopically labelled compounds
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