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Motivation: Liquid chromatography-mass spectrometry (LC/MS) is a key technique in metabolomics. Since the efficient assignment of MS signals to true biological metabolites becomes feasible in combination with in vivo stable isotopic labelling, our aim was to provide a new software tool for this purpose., Results: An algorithm and a program (MetExtract) have been developed to search for metabolites in in vivo labelled biological samples. The algorithm makes use of the chromatographic characteristics of the LC/MS data and detects MS peaks fulfilling the criteria of stable isotopic labelling. As a result of all calculations, the algorithm specifies a list of m/z values, the corresponding number of atoms of the labelling element (e.g. carbon) together with retention time and extracted adduct-, fragment- and polymer ions. Its function was evaluated using native (12)C- and uniformly (13)C-labelled standard substances., Availability: MetExtract is available free of charge and warranty at http://code.google.com/p/metextract/. Precompiled executables are available for Windows operating systems., Supplementary Information: Supplementary data are available at Bioinformatics online.

Methods for the determination of toxic pyrrolizidine alkaloids in plants and foods are described with emphasis on the important aspects of sample extraction and clean-up and the now preferred determination by liquid chromatography-mass spectrometry. The efficiencies of different extraction solvents and methods are described, as are the methods of reduction of N-oxides. Appropriate liquid chromatography-mass spectrometry conditions are tabulated. This concise review is intended to guide analysts towards adopting a more unified and reliable approach to the analysis of these important toxins.

About 300-400 mycotoxins are known today. To some extent these compounds show very different physicochemical properties, which led to a vast quantity of analytical methods for single toxins or certain classes of mycotoxins in a variety of matrices. Due to synergistic effects of co-occurring toxins, endeavors have been made to simultaneously detect and quantify several classes of mycotoxins. This paper discusses several of the published LC-MS/MS multi-mycotoxin-methods and also introduces a new method, which allows the concurrent detection and quantification of 90 major mycotoxins and other secondary fungal metabolites in cereals. Even more, known plant derived metabolites of mycotoxins, like zearalenone-4-glucoside or deoxynivalenol-3-glucoside are included in this method. The significance of mycotoxin conjugates is briefly discussed as well.

In this paper, the structure and the identity of fully 13C-substituted T-2 toxin were confirmed using high-resolution mass spectrometry, 1H-NMR, 13C-NMR, tandem mass spectrometry and HPLC-DAD. The purity of this compound was estimated to be at least 98.8% according to UV data. The isotopic distribution of (13C(24)) T-2 toxin indicated a total isotopic enrichment of 98.2 +/- 1.0 atom% 13C, and the application of different MS measurement modes revealed the MS/MS fragmentation pattern of T-2 toxin. Furthermore, a stable isotope dilution mass spectrometry method for the quantification of T-2 toxin was developed using (13C(24)) T-2 toxin as internal standard. The method was evaluated with and without conventional clean-up and validated for maize and oats. Both cereals showed strong matrix enhancement effects, which could be compensated for through the application of the isotope-substituted internal standard.

A novel method for the simultaneous determination of the Fusarium mycotoxins nivalenol, deoxynivalenol, fusarenon-X, 3-acetyl-deoxynivalenol, the sum of 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, diacetoxy-scirpenol, HT-2 toxin, T-2 toxin and zearalenone in maize has been developed using gradient RP-LC with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC-APCI-MS/MS). Swift clean-up of maize samples was performed with MycoSep #226 columns. Quantification of zearalenone was performed with zearalanone as internal standard (IS), while no IS was used for the trichothecenes. Detection of the mycotoxins was carried out in the multiple reaction monitoring (MRM) mode. Method performance characteristics were estimated after analysis of spiked blank maize samples. Calibration curves were linear between 10 and 1000 microg/kg and the limits of detection ranged from 0.3 to 3.8 microg/kg depending on the mycotoxin. Moreover, the accuracy of the method was confirmed by comparing analytical data to certified values from reference materials for deoxynivalenol and zearalenone.

Glucosides of several Fusarium mycotoxins occur in naturally infected cereals and may contribute to an increased content to the total mycotoxin load of food and feed. The paper presents the results of a fermentation procedure to produce zearalenone-4O-β-D-glucopyranoside from zearalenone using an engineered Saccharomyces cerevisiae strain, expressing the Arabidopsis thaliana UDP-glucosyltransferase UGT73C6. About 24 mg of zearalenone-4O-β-D-glucopyranoside was obtained from 50 mg of zearalenone and further purified. A total of 10 mg of the glucoside were reduced with sodium borohydride, yielding 4.1 mg α-zearalenol-4O-β-D-glucopyranoside and 4.5 mg β-zearalenol-4O-β-D-glucopyranoside at purities higher than 99%. To confirm the identities of the three produced glucosides, MS and MS/MS spectra were acquired using negative electrospray ionization. Besides the deprotonated ions at m/z 479 or 481, respectively, in full-scan mode, fragments, adducts, and dimers were recorded and assigned. MS/MS spectra of the glucosylated substances yielded the deprotonated ions of the mycotoxins zearalenone, α-zearalenol, β-zearalenol and their fragments, respectively. Unambiguous structural assignment of the three substances was achieved using two-dimensional NMR methods. This way, the glucose attachment to position C-4, the β-configuration of the sugar unit and the stereo-chemical assignment of the zearalenol hydroxyl group at C-6' were proven. [ABSTRACT FROM AUTHOR]

In this paper, the structure and the identity of fully 13C-substituted T-2 toxin were confirmed using high-resolution mass spectrometry, 1H-NMR, 13C-NMR, tandem mass spectrometry and HPLC–DAD. The purity of this compound was estimated to be at least 98.8% according to UV data. The isotopic distribution of (13C24) T-2 toxin indicated a total isotopic enrichment of 98.2 ± 1.0 atom% 13C, and the application of different MS measurement modes revealed the MS/MS fragmentation pattern of T-2 toxin. Furthermore, a stable isotope dilution mass spectrometry method for the quantification of T-2 toxin was developed using (13C24) T-2 toxin as internal standard. The method was evaluated with and without conventional clean-up and validated for maize and oats. Both cereals showed strong matrix enhancement effects, which could be compensated for through the application of the isotope-substituted internal standard. [ABSTRACT FROM AUTHOR]

Plants have a high capacity to transform and thereby detoxify deleterious or poisonous compounds, like mycotoxins. The formation of glucose conjugates has a central role in this process. Mammals, however, are able to (partly) release the precursor substances during digestion, reactivating the mycotoxins. This short review provides a brief summary about the metabolism of the Fusarium mycotoxins deoxynivalenol and zearalenone in plants. Two examples are discussed in greater detail. First, the formation of deoxynivalenol-3-glucoside in wheat is linked to a quantitative trait locus that is often used for Fusarium head blight resistance breeding. Secondly, the metabolism of zearalenone in Arabidopsis thaliana results in at least 17 different metabolites, all of which are potentially hazardous for humans and animals. [ABSTRACT FROM AUTHOR]

The biotransformation products of zearalenone, a Fusarium mycotoxin, were elucidated using the model plant Arabidopsis thaliana. After treatment of plant seedlings with 50 µM zearalenone, both the liquid media and the plant extracts were analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). An array of 17 different metabolites, most prominently glucosides, malonylglucosides, di-hexose- and hexose–pentose disaccharides of zearalenone, and α- and β-zearalenol, were detected in the samples. Time courses for the different zearalenone metabolites were recorded and they give a closer insight into the metabolism kinetics. A scheme proposing the zearalenone metabolism in A. thaliana is given. The aspect of food safety regarding the (potential) occurrence of masked mycotoxins in agricultural commodities is discussed. [ABSTRACT FROM AUTHOR]

The powerful combination of liquid chromatography and mass spectrometry (MS) is often limited by matrix effects during ionization in the MS ion source. The use of fully isotope-substituted (13C15)-deoxynivalenol ((13C15)-DON) as an internal standard (IS) corrects matrix effects and improves the accuracy of analytical methods using mass spectrometry for the quantitative determination of the Fusarium mycotoxin deoxynivalenol (DON). The IS was characterized with respect to its chromatographic purity by liquid chromatography-ultraviolet light and its isotope distribution by time-of-flight mass spectrometry. Its low-energy collision-induced dissociation behaviour was compared with DON. Moreover, this work describes the successful application of (13C15)-DON as IS for the determination of DON in maize using high-performance liquid chromatography (HPLC) electrospray (ESI) with tandem mass spectrometry. The results demonstrate that the IS can successfully correct for fluctuations during extraction and clean-up of the sample as well as the ionization of DON in the MS ion source. Random variations in ionization affect the IS in the same way as the analyte. Recoveries for DON in maize of 76% ± 1.9% (external calibration) or 101% ± 2.4% (internal calibration) were reached, respectively, after sample clean-up. [ABSTRACT FROM AUTHOR]

Very often, the accuracy of quantitative analytical methods for the determination of mycotoxins by liquid chromatography (LC)-mass spectrometry (MS) and LC-MS/MS is limited by matrix effects during the ionization process in the MS source. Stable isotope labeled standards are best suited to correct for matrix effects and to improve both the trueness and the precision of analytical methods employing LC-MS and LC-MS/MS. This paper describes the successful use of fully 13C isotope labeled deoxynivalenol [(13C15)DON] as an internal standard (IS) for the accurate determination of DON in maize and wheat by LC electrospray ionization MS/MS. To show the full potential of (13C15)DON as IS, maize and wheat extracts were analyzed without further cleanup. Subsequent to calibration for the LC-MS end determination, DON was quantified in matrix reference materials (wheat and maize). Without consideration of the IS, apparent recoveries of DON were 29±6% ( n=7) for wheat and 37±5% ( n=7) for maize. However, the determination of DON in the reference materials yielded 95±3% (wheat) and 99±3% (maize) when (13C15)DON was used as an IS for data evaluation. [ABSTRACT FROM AUTHOR]

The lack of reliable, certified calibrant solutions for the Fusarium mycotoxins deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON) and nivalenol (NIV) is a serious drawback in the already problematic area of trichothecene analysis. For this reason, purified DON, 3-Ac-DON, 15-Ac-DON and NIV standards were processed, the conditions required for their isolation and purification were optimised, and the crystalline toxins were thoroughly characterised. Several complimentary analytical methods were used to evaluate the identities of the mycotoxins and the types and amounts of impurities; results obtained from 1 H and 13 C NMR spectra, as well as from IR-spectra, were in agreement with the literature. Elemental analysis revealed that the isolated NIV occurs as monohydrate. If this is not known it results in a weighing error of approximately 5%. Differential scanning calorimetry (DSC) was only successful for 15-Ac-DON, as the other trichothecenes decomposed during measurements. No traces of chloride, nitrate and sulphate were found by means of ion chromatography (IC). As expected UV absorption spectra for DON, NIV, 3-Ac-DON and 15-Ac-DON yielded λmax values of 216, 217, 217 and 219 nm, respectively. Minor peaks due to impurities were observed by high performance liquid chromatography (HPLC) with UV detection. The main impurity peak in the DON sample was identified by LC-tandem mass spectroscopy (LC-MS/MS) as 4,7-dideoxy-NIV (7-deoxy-DON), which occurs at levels of approximately 1.4%. Gas chromatography (GC) was performed, coupled with either an electron capture detector (ECD), a flame ionisation detector (FID), or a mass spectrometric detector (MS); however, derivatisation prior to GC analysis makes the estimation of impurities difficult. LC-MS/MS was found to be unsuitable for quantifying levels of impurities. It can be concluded that high-purity (>97%) B-trichothecene standards were successfully processed and fully characterised for the first time. [ABSTRACT FROM AUTHOR]