Table of Lectures - Society for Mycotoxin Research

L-02 29 th Mycotoxin Workshop
Development of a multi-stable isotope dilution assay (SIDA) for
the determination of trichothecenes in food commodities
Stefan Asam, Michael Rychlik
Lehrstuhl für Lebensmittelchemie der Technischen Universität München,
Lichtenbergstrasse 4, D-85748 Garching, Germany
Trichothecenes are regularly occurring in cereals and cereal products. This contamination may include
both the very polar type B trichothecenes and the more nonpolar type A trichothecenes such as
deoxynivalenol and T2-toxin, respectively. Thus the development of methods for multicomponent analysis
of substances with decisively unequal physical-chemical properties require certain compromise in
terms of extraction solvent, clean-up and chromatographic conditions. In consequence, differences in
the efficiency of the extraction procedure, in analyte losses during clean-up and ion suppression in the
ESI-interface during LC-MS/MS-analysis may occur, which altogether account for the recovery of the
analyte. To avoid laborious and time-consuming method optimation and validation, the application of
stable isotope labelled internal standards is generally recognised as the method of choice.
Therefore, we synthesized several labelled type A and type B trichothecenes and developed respective
stable isotope dilution assays. Synthesis of [13C2]-15-acetyldeoxynivalenol, [13C2]-3-acetyldeoxynivalenol,
[13C2]-4-acetylnivalenol, [13C4]-T2-toxin, [13C2]-HT2-toxin, [13C4]-diacetoxyscirpenol and
[13C2]-15-monoacetoxyscirpenol was accomplished by [13C2]-acetylation of the corresponding parent
alcohol, i.e. deoxynivalenol, nivalenol, T2-triol and scirpentriol. The resulting peracetylated compounds
were selectively hydrolyzed to obtain the desired mono- and diacetates. All synthesized products were
characterized by NMR and MS experiments. [13C15]-deoxynivalenol was purchased commercially.
By using the synthesized labelled trichothecenes and [13C15]-deoxynivalenol stable isotope dilution assays
were developed. Food samples were spiked with internal standards, extracted with common extraction
mixtures and purified by non retentative SPE-columns of different manufacturers. All trichothecenes
under study were quantified simultaneously within one LC-MS run using single and tandem MS detection.
The method revealed good sensitivity with low detection and quantification limits along with excellent
recovery data and good precision in inter-assay studies. A series of food samples was analysed showing
a frequent contamination of cereals with deoxynivalenol at a medium concentration level mostly lower
than the regulatory limit in the EU. Besides deoxynivalenol larger amounts of 15-acetyldeoxynivalenol
were detected in maize and processed maize snacks, which is critical due to the increased toxicity of
the acetylated trichothecenes. T2-toxin and HT2-toxin were regularly found in oat and oat products with
the sum of both contents generally exceeding 50 µg/kg.
Whereas the clean-up procedure using non retentative SPE-columns had no major effect on the recovery
of the trichothecenes, the polar deoxynivalenol especially was incompletely extracted by the common
extraction solvents, which are composed of at least 60 % organic solvent. Though T2-toxin was completely
extracted quantitation using external calibration resulted in lower values than the SIDA method,
which points to a certain ion suppressing effect of coeluting matrix components. As a consequence
the use of stable isotope labelled internal standards is an essential tool to obtain accurate results for
evaluating trichothecene contamination of food commodities.
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