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

Meng-Reiterer J, Varga E, Nathanail AV, Bueschl C, Rechthaler J, McCormick SP, Michlmayr H, Malachová A, Fruhmann P, Adam G, Berthiller F, Lemmens M, Schuhmacher R - Anal Bioanal Chem (2015)

Bottom Line: 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.Graphical Abstract Isotope-assisted untargeted screening of HT-2 toxin and T-2 toxin metabolites in barley.

View Article: PubMed Central - PubMed

Affiliation: Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad-Lorenz-Str. 20, 3430, Tulln, Austria.

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 Abstract Isotope-assisted untargeted screening of HT-2 toxin and T-2 toxin metabolites in barley.

No MeSH data available.


Related in: MedlinePlus

Relative time courses of HT-2 toxin-derived metabolites. Barley ears were treated with 200 μg HT-2 toxin and harvested immediately, 1, 3 and 7 days after treatment and at full-ripening stage. Relative amounts (areas of extracted ion chromatogram peaks normalised by ear weight) are plotted versus harvest time point after treatment. Analysis was performed with a 6550 iFunnel Q-TOF LC/MS system. Error bars refer to the standard deviation of biological triplicates. HT2 HT-2 toxin, T2 T-2 toxin, Glc glucoside, MalGlc malonylglucoside
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Fig5: Relative time courses of HT-2 toxin-derived metabolites. Barley ears were treated with 200 μg HT-2 toxin and harvested immediately, 1, 3 and 7 days after treatment and at full-ripening stage. Relative amounts (areas of extracted ion chromatogram peaks normalised by ear weight) are plotted versus harvest time point after treatment. Analysis was performed with a 6550 iFunnel Q-TOF LC/MS system. Error bars refer to the standard deviation of biological triplicates. HT2 HT-2 toxin, T2 T-2 toxin, Glc glucoside, MalGlc malonylglucoside

Mentions: Regarding HT2, approximately 53 % (0.250 ± 0.054 μmol) was transformed to HT2-3-O-β-Glc, whilst 25 % (0.116 ± 0.033 μmol) remained unmodified as parent toxin within the first 24 h upon treatment. With increasing time, a decrease of HT2 and HT2-3-O-β-Glc was observed which ended in a content of 7 % (0.033 ± 0.007 μmol) and 34 % (0.161 ± 0.034 μmol) relative to the originally added HT2 after ripening, respectively. This finding confirms that HT2-3-O-β-Glc is further metabolised and correlates well with the relative quantification (Fig. 5) of HT2 metabolites. Taking a closer look at the formation of HT2 biotransformation products over time, hydroxy-HT2-Glc, hydroxy-HT2-MalGlc, HT2-di-Glc, HT2-MalGlc, as well as T2-triol-Glc (detectable only at one time point) show maximal abundance after ripening, leading to the assumption that they are derived from early formed HT2-3-O-β-Glc. Moreover, 15-acetyl-T2-tetraol-Glc and 15-acetyl-T2-tetraol-MalGlc were also found to be produced after 1 day. Although absolute quantification was not possible for HT2-MalGlc due to the lack of an authentic standard, comparison of EIC peak areas suggests that it belongs to the major biotransformation products.Fig. 5


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

Meng-Reiterer J, Varga E, Nathanail AV, Bueschl C, Rechthaler J, McCormick SP, Michlmayr H, Malachová A, Fruhmann P, Adam G, Berthiller F, Lemmens M, Schuhmacher R - Anal Bioanal Chem (2015)

Relative time courses of HT-2 toxin-derived metabolites. Barley ears were treated with 200 μg HT-2 toxin and harvested immediately, 1, 3 and 7 days after treatment and at full-ripening stage. Relative amounts (areas of extracted ion chromatogram peaks normalised by ear weight) are plotted versus harvest time point after treatment. Analysis was performed with a 6550 iFunnel Q-TOF LC/MS system. Error bars refer to the standard deviation of biological triplicates. HT2 HT-2 toxin, T2 T-2 toxin, Glc glucoside, MalGlc malonylglucoside
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4595538&req=5

Fig5: Relative time courses of HT-2 toxin-derived metabolites. Barley ears were treated with 200 μg HT-2 toxin and harvested immediately, 1, 3 and 7 days after treatment and at full-ripening stage. Relative amounts (areas of extracted ion chromatogram peaks normalised by ear weight) are plotted versus harvest time point after treatment. Analysis was performed with a 6550 iFunnel Q-TOF LC/MS system. Error bars refer to the standard deviation of biological triplicates. HT2 HT-2 toxin, T2 T-2 toxin, Glc glucoside, MalGlc malonylglucoside
Mentions: Regarding HT2, approximately 53 % (0.250 ± 0.054 μmol) was transformed to HT2-3-O-β-Glc, whilst 25 % (0.116 ± 0.033 μmol) remained unmodified as parent toxin within the first 24 h upon treatment. With increasing time, a decrease of HT2 and HT2-3-O-β-Glc was observed which ended in a content of 7 % (0.033 ± 0.007 μmol) and 34 % (0.161 ± 0.034 μmol) relative to the originally added HT2 after ripening, respectively. This finding confirms that HT2-3-O-β-Glc is further metabolised and correlates well with the relative quantification (Fig. 5) of HT2 metabolites. Taking a closer look at the formation of HT2 biotransformation products over time, hydroxy-HT2-Glc, hydroxy-HT2-MalGlc, HT2-di-Glc, HT2-MalGlc, as well as T2-triol-Glc (detectable only at one time point) show maximal abundance after ripening, leading to the assumption that they are derived from early formed HT2-3-O-β-Glc. Moreover, 15-acetyl-T2-tetraol-Glc and 15-acetyl-T2-tetraol-MalGlc were also found to be produced after 1 day. Although absolute quantification was not possible for HT2-MalGlc due to the lack of an authentic standard, comparison of EIC peak areas suggests that it belongs to the major biotransformation products.Fig. 5

Bottom Line: 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.Graphical Abstract Isotope-assisted untargeted screening of HT-2 toxin and T-2 toxin metabolites in barley.

View Article: PubMed Central - PubMed

Affiliation: Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad-Lorenz-Str. 20, 3430, Tulln, Austria.

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 Abstract Isotope-assisted untargeted screening of HT-2 toxin and T-2 toxin metabolites in barley.

No MeSH data available.


Related in: MedlinePlus