Isotopic labeling-assisted metabolomics using LC-MS.

Metabolomics has emerged as the latest of the so-called "omics" disciplines and has great potential to provide deeper understanding of fundamental biochemical processes at the biological system level. Among recent technological developments, LC-HRMS enables determination of hundreds to thousands of metabolites over a wide range of concentrations and has developed into one of the most powerful techniques in non-targeted metabolomics. The analysis of mixtures of in-vivo-stable isotopic-labeled samples or reference substances with un-labeled samples leads to specific LC-MS data patterns which can be systematically exploited in practically all data-processing steps. This includes recognition of true metabolite-derived analytical features in highly complex LC-MS data and characterization of the global biochemical composition of biological samples. In addition, stable-isotopic labeling can be used for more accurate quantification (via internal standardization) and identification of compounds in different organisms.

Evaluation of LC-high-resolution FT-Orbitrap MS for the quantification of selected mycotoxins and the simultaneous screening of fungal metabolites in food.

A liquid chromatography-high-resolution mass spectrometry-based method is reported for the quantification of 20 selected mycotoxins and the simultaneous screening for 200 fungal metabolites in food. For regulated mycotoxins, such as aflatoxins, fumonisins, ochratoxin A, zearalenone and trichothecenes, the evaluation of the method performance characteristics, such as precision, trueness, limit of detection and matrix effects, has been exemplified for the matrix maize. In the case of the limit of detection, an alternative evaluation approach for high-resolution FT-Orbitrap data is proposed. Measurements of the signal-to-noise ratios obtained from 'full-profile mode' data led to detection limits between 8 and 160 ng g(-1). Eight naturally contaminated wheat- and maize-based matrix test materials, originating from interlaboratory comparison studies, were used to confirm the trueness of the method for deoxynivalenol, zearalenone, fumonisin B(1) and B(2), HT-2, and T-2 toxin. In addition to accurate quantification of the most relevant mycotoxins, the full-scan chromatograms were used to investigate the potential of the FT-Orbitrap to screen simultaneously for a large number of fungal metabolites. First, a list of 200 metabolites, potentially being present in food samples, was established. Next, specific detection and identification criteria were defined, which are based on accurate mass, peak intensity and isotopologue ratio. The application of these criteria to the suspected metabolites from the list resulted in the putative identification of 13 fungal metabolites in addition to the target toxins.

Preparation and characterization of the conjugated Fusarium mycotoxins zearalenone-4O-beta-D-glucopyranoside, alpha-zearalenol-4O-beta-D-glucopyranoside and beta-zearalenol-4O-beta-D-glucopyranoside by MS/MS and two-dimensional NMR.

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-beta-D-glucopyranoside from zearalenone using an engineered Saccharomyces cerevisiae strain, expressing the Arabidopsis thaliana UDP-glucosyltransferase UGT73C6. About 24 mg of zearalenone-4O-beta-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 alpha-zearalenol-4O-beta-D-glucopyranoside and 4.5 mg beta-zearalenol-4O-beta-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, alpha-zearalenol, beta-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 beta-configuration of the sugar unit and the stereo-chemical assignment of the zearalenol hydroxyl group at C-6' were proven.

Occurrence of deoxynivalenol and its 3-beta-D-glucoside in wheat and maize.

Deoxynivalenol-3-beta-D-glucoside (D3G), a phase II plant metabolite of the mycotoxin deoxynivalenol (DON), occurs in naturally Fusarium-contaminated cereals. In order to investigate the frequency of occurrence as well as the relative and absolute concentrations of D3G in naturally infected cereals, 23 wheat samples originating from fields in Austria, Germany and Slovakia as well as 54 maize samples from Austrian fields were analysed for DON and D3G by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both analytes were detected in all the 77 field samples. DON was found at levels from 42 to 4130 ng g(-1) (977 +/- 1000 ng g(-1) on average). The D3G concentrations in all cereal samples were in the range 10-1070 ng g(-1) (216 +/- 253 ng g(-1) on average), corresponding to about 5-46 mol% of their DON concentrations (15 +/- 8 mol% on average).

Characterization of (13C24) T-2 toxin and its use as an internal standard for the quantification of T-2 toxin in cereals with HPLC-MS/MS.

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.

Short review: Metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants.

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 theFusarium 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 forFusarium head blight resistance breeding. Secondly, the metabolism of zearalenone inArabidopsis thaliana results in at least 17 different metabolites, all of which are potentially hazardous for humans and animals.

Production of zearalenone-4-glucoside, a-zearalenol-4-glucoside and ß-zearalenol-4-glucoside.

The work at hand describes the production of the zearalenone (ZON) metabolites zearalenone-4-glucoside (ZON-4G), a-zearalenol-4-glucoside (oc-ZOL-4G) and ß-zearalenol-4-glucoside (ß-ZOL-4G). In a first step a genetically modified yeast strain, expressing theArabidopsis thaliana UDP-glu-cosyltransferase UGT73C6, was treated with ZON to produce ZON-4G. The substance was purified by solid phase extraction and subsequent reversed phase preparative HPLC prior to the reduction with sodium borohydride to yield 0C-ZOL-4G and ß-ZOL-4G. The identity and purity of the substances were confirmed by(13)C-and(1)H-NMR as well as by HPLC-UV. In total, 50 mg of ZON were used to produce 5 mg of a-ZOL-4G with a purity of 98%, 6 mg of ß-ZOL-4G with a purity of 99% and 5 mg of ZON-4G with a purity of 99%.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) determination of phase II metabolites of the mycotoxin zearalenone in the model plant Arabidopsis thaliana.

The biotransformation products of zearalenone, a Fusarium mycotoxin, were elucidated using the model plant Arabidopsis thaliana. After treatment of plant seedlings with 50 microM 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 alpha- and beta-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.

Characterization and application of isotope-substituted (13C15)-deoxynivalenol (DON) as an internal standard for the determination of DON.

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.

Suitability of a fully 13C isotope labeled internal standard for the determination of the mycotoxin deoxynivalenol by LC-MS/MS without clean up.

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.

Processing and purity assessment of standards for the analysis of type-B trichothecene mycotoxins.

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 1H and 13C 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 lambda(max) 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.

First results of GEN-AU: Cloning of Deoxynivalenol- and Zearalenone-inactivating UDP-glucosyltransferase genes fromArabidopsis thaliana and expression in yeast for production of mycotoxin-glucosides.

First results of the GEN-AU pilot project "Fusarium virulence and plant resistance mechanisms" are reported. Employing genetically engineered yeast strains we have been able to clone genes from the model plantArabidopsis thaliana encoding UDP-glucosyltransferases which can inactivate deoxynivalenol (DON) and zearalenone (ZON). The structure of the metabolites produced by the transformed yeast strains were determined by LC-MS/MS as DON-3O-glucoside and ZON-4O-glucoside, respectively. ZON and derivatives added to glucosyltransferase expressing yeast cultures are converted into the corresponding glucosides in very high yield, opening an efficient way to produce reference materials for these masked mycotoxins.

[Determination of DON-3-Glucoside in artificially and naturally contaminated wheat with LC-MS/MS]. / Bestimmung von DON-3-Glukosid in künstlich und natürlich kontaminiertem Weizen mittels LC-MS/MS.

Naturally contaminated and artificiallyFusarium spp. inoculated wheat was analyzed for deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol. After extraction and clean-up with MycoSep columns, the trichothecenes were determined using a LC-ESI-MS/MS method. Deoxynivalenol-3-glucoside was detectable in 4 out of 4 artificially inoculated and in 22 out of 25 naturally contaminated wheat samples. For the latter, the average relative deoxynivalenol-3-glucoside concentration was about 6% of the deoxynivalenol concentration. The maximum relative deoxynivalenol-3-glucoside concentration was 12% as compared to the concentration of deoxynivalenol. In all samples, the concentration of deoxynivalenol-3-glucoside was higher than the concentrations of 3-acetyl-deoxynivalenol or 15-acetyl-deoxynivalenol.

Simultaneous determination of type A-& B-trichothecenes and zearalenone in cereals by High Performance Liquid Chromatography - Tandem Mass Spectrometry.

A rapid quantitative method for the simultaneous determination of the majorFusarium mycotoxins nivalenol, deoxynivalenol, fusarenon-X, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, diacetoxyscirpenol, HT-2 toxin, T-2 toxin and zearalenone in maize and wheat was developed. Raw extracts (acetonitrile/water 84/16) are cleaned-up with MycoSep(®) columns., Chromatographic separation and end determination is carried out by HPLC-APCI-MS/MS.HPLC run times of 10 minutes considerably increases sample throughput and make this method suitable for routine analysis. The use of a triple quadrupole mass spectrometer allows the selective detection of the mycotoxins and their quantification in the low µg/kg-range.

Performance of new clean-up column for the determination of ochratoxin A in cereals and foodstuffs by HPLC-FLD.

The performance of the newly developed Mycosep 229 Ochra and Multisep 229 Ochra clean-up columns for ochratoxin A (OTA) determination was evaluated. OTA was subsequently analysed using RP-HPLC with fluorescence detection. Recoveries for frequently contaminated commodities, like cereals, red wine, raisins and green coffee, were estimated. The recoveries obtained for the Mycosep 229 Ochra column were in the range from 87.9 +/- 12.5% (n = 6) for wheat to 99.4 +/- 2.7% (n = 24) for raisins. For Multisep 229 Ochra, recoveries from 76.5 +/- 8.0% (n = 6) for barley to 86.4 +/- 1.4% (n = 24) for raisins were achieved. Limits of detection for all matrices investigated (maize, wheat, rice, barley, raisins, green coffee beans, red wine) were in the range 0.4-2.4 microg kg(-1). The trueness of the method was tested using a certified reference material.

Synthesis of deoxynivalenol-glucosides and their characterization using a QTrap LC-MS/MS.

Deoxynivalenol (DON)-glucosides were successfully synthesized in a two-step reaction from 1-ß-Bromo-1-deoxy-2,3,4,6-tetra-O-acetyl-α-D-gluco-pyranose and 3-Acetyl-DON or 15-Acetyl-DON. After purification of the reaction products, the mycotoxin conjugates were for the first time characterized by means of a triple quadrupole mass spectrometer in combination with a linear ion trap. Due to different fragmentation behaviour it was also possible to distinguish between the two glucosides.

International interlaboratory study for the determination of the Fusarium mycotoxins zearalenone and deoxynivalenol in agricultural commodities.

Twenty-eight laboratories from 12 different countries participated in an interlaboratory study for the determination of the Fusarium mycotoxin zearalenone (ZON) in maize and deoxynivalenol (DON) in maize and wheat employing their usual in-house methods. The aim of this study was to obtain information about the state-of-the-art of ZON and DON analysis in cereals and to support a knowledge and experience exchange between the participating laboratories in the field of mycotoxin analysis. Eight different sample types were distributed to the participants, 'blank' materials, spiked samples (102 microg/kg ZON in maize and 475 microg/kg DON in wheat) and naturally-contaminated maize and wheat. For the final separation and quantification either gas chromatography (GC), high performance liquid chromatography (HPLC), thin layer chromatography (TLC) or enzyme linked immunosorbent assays (ELISA) were employed by the participating laboratories. Coefficients of variation (CV) between laboratory mean results (outliers rejected) ranged from 28 to 41% for ZON and from 32 to 38% for DON. The results are close to the between laboratory CV criteria of 40% for DON and ZON at concentration levels of >100 microg/kg established by the CEN in 1999. A good trueness was obtained for the wheat samples spiked at 475 microg/kg DON. However, a significant deviation at p = 0.01 from the respective target value was observed for the maize samples spiked at 102 microg/kg ZON. The high CVs can be traced back to problems occurring by determination of the concentration of the participants' own calibrant solutions. Additionally, the variability of the results is strongly influenced by the use of different final separation and quantification procedures.

Determination of measurement uncertainty for the determination of triazines in groundwater from validation data.

Laboratories are increasingly urged to submit full uncertainties of their analytical results rather than only standard deviations. The determination of measurement uncertainties in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM) is demonstrated using the validation approach explicitly endorsed by the recent edition of the EURACHEM guide for the determination of measurement uncertainty. Measurement uncertainty was split into uncertainty of the sample mass, uncertainty of the concentration of the stock standard solution, uncertainty of the calibration and uncertainty connected to within- and between-series precision. Uncertainties of sample mass and of the concentration of the stock standard solution were 0.26 and 1.14% for all analytes, which is negligible compared with the contributions of precision and calibration. Uncertainty of calibration was estimated from the calibration graph. Relative uncertainty of calibration was found to be strongly concentration dependent and to be the main uncertainty contribution below 0.2 microgram L-1. Precision was split into within-series and between-series standard deviation, which dominate the combined standard uncertainty at higher concentrations. The results obtained from these calculations are compared with results for a certified reference material and with the performance in an interlaboratory comparison. It was found that all results agreed within their uncertainty with the target values, showing that the estimated uncertainties are realistic.

Effects of beauvericin to mammalian tissue and its production by Austrian isolates ofFusarium proliferatum and Fusarium subglutinans.

Austrian isolates ofFusarium subglutinans andFusarium proliferatum were studied for their ability to produce beauvericin, moniliformin and fumonisin B1 and B2 under laboratory conditions. Analytical methodology for beauvericin was specially adapted for this task. Our analyses showed that the strains produced beauvericin up to 687 mg /kg maize and moniliformin up to 70 mg/kg. The culture ofF. proliferatum in addition produced fumonisin B1 and B2 at levels of 106 and 61 mg/kg,respectively. The preliminary toxicity experiments performed in this study clearly indicated a toxic effect of beauvericin on the contractility of mammalian smooth muscle and thus on mammalian cells.

Determination of the Fusarium mycotoxin beauvericin at micrograms/kg levels in corn by high-performance liquid chromatography with diode-array detection.

A method is described for the detection of the Fusarium mycotoxin beauvericin (BEA) in corn and corn meal. Spectral data obtained with a diode-array detector showed that the most sensitive wavelength for the detection of BEA is 192 nm. The detection limit for BEA was 50 micrograms/kg, which is an increase in sensitivity by a factor of at least twenty compared to previously published analytical methods for this mycotoxin.