Deoxynivalenol (DON) is a mycotoxin produced by members of the Fusarium species. Contamination by DON has been found in cereal grains, in particular wheat, barley, oats, corn and rice. Besides other effects, this toxin can cause intestinal upset and flu-like symptoms if it is ingested.1 Identifying new tools and methods to accurately detect DON and other xenobiotics in wheat and other cereal grains are of interest to researchers and cereal grain farmers alike.
Kluger et al. recently developed an untargeted screening strategy for detecting DON using stable isotopic labeling (SIL) and liquid chromatography–high resolution mass spectrometry (LC-HRMS)2. For their experiments, the researchers used lyophilized non-labelled DON mixed 1+1 with 13C15-labelled DON as the DON inoculation solutions.
The researchers grew wheat plants (cultivar “Remus”) in a controlled environment and treated them with either DON inoculation solution or water (as “mock-treated” plants). They treafirst treated the plants by injecting them with either 10 μL of DON or water into each of two adjacent spikelets in the lower part of a flowering ear. Twenty-four hours later, the next two adjacent spikelets, located immediately above those previously treated, were treated in the same manner. They repeated this procedure (in triplicate) after 48, 72 and 96 hours. The researchers separated the DON-treated and the mock-treated ears after 108 hours had passed since the first inoculation. The separated ears were then shock-frozen in liquid nitrogen and milled into a fine powder in preparation for mass spectrometry.
The LC-HR-MS measurements were performed on an LTQ Orbitrap XL equipped with an electrospray ionization source coupled to an UHPLC system. Data were recorded and evaluated using Xcalibur 2.1 (all from Thermo Scientific).
The researchers used an improved software program called MetExtract to extract corresponding MS peak pairs that were of interest. The software required the DON-metabolization products meet three characteristics to be considered a positive identific
ation. First, the DON metabolization products had to contain monoisotopic non-labelled and completely labelled isotopologs of DON-derivative ions and, these ions needed to form the principal ions of their corresponding isotopic patterns. Secondly, the software required that the peak area of monoisotopic peaks and fully labelled analogs was between 0.8-1.2. Lastly, the monoisotopic non-labelled and the completely labelled isotopologs ions needed to show chromatic co-elution. The metabolites were characterized if the accurate mass differed 5 ppm from the theoretical mass, and heteroatoms present within the conjugates were determined by evaluating fully labelled isotopologs.
MetExtract revealed 57 ion pairs in full-scan chromatograms of DON-treated samples. The monoisotopic non-labelled and the corresponding completely labelled isotopolog of the DON moiety were shown in these samples, while no ion pairs were detected in mock-treated samples.
Interestingly, the researchers found that the DON metabolites were intact, and DON degradation products all had at least 15 carbon atoms, which meant that they contained the intact DON carbon skeleton. They also found structurally different DON conjugates that contained a molecular weight that was much higher than expected. Moreover, the researchers also confirmed the presence of eight biotransformation products, including: DON-3-glucoside, DON-glutathione, DON-S-cysteine, DON-S-cysteinyl-glycine. There were also five unknown conjugates that will require additional experiments to identify. As a whole, the authors consider this work contains a high potential for future applications in the metabolization of other xenobiotics.
1. Sobrova, P., et al. (2010) “Deoxynivalenol and its toxicity.”, Interdisciplinary Toxicology, Sep 2010; 3(3): (pp. 94–99), Published online Sep 2010. doi: 10.2478/v10102-010-0019-x PMCID: PMC2984136
2. Kluger B. et al. (2013) “Stable isotopic labelling-assisted untargeted metabolic profiling reveals novel conjugates of the mycotoxin deoxynivalenol in wheat.”, Analytical and Bioanalytical Chemistry, 405(15) (pp. 5031-6) doi: 10.1007/s00216-012-6483-8.