Robert Köppen

Determination of mycotoxins in foods: current state of analytical methods and limitations

Mycotoxins are natural contaminants produced by a range of fungal species. Their common occurrence in food and feed poses a threat to the health of humans and animals. This threat is caused either by the direct contamination of agricultural commodities or by a “carry-over” of mycotoxins and their metabolites into animal tissues, milk, and eggs after feeding of contaminated hay or corn. As a consequence of their diverse chemical structures and varying physical properties, mycotoxins exhibit a wide range of biological effects. Individual mycotoxins can be genotoxic, mutagenic, carcinogenic, teratogenic, and oestrogenic. To protect consumer health and to reduce economic losses, surveillance and control of mycotoxins in food and feed has become a major objective for producers, regulatory authorities and researchers worldwide. However, the variety of chemical structures makes it impossible to use one single technique for mycotoxin analysis. Hence, a vast number of analytical methods has been developed and validated. The heterogeneity of food matrices combined with the demand for a fast, simultaneous and accurate determination of multiple mycotoxins creates enormous challenges for routine analysis. The most crucial issues will be discussed in this review. These are (1) the collection of representative samples, (2) the performance of classical and emerging analytical methods based on chromatographic or immunochemical techniques, (3) the validation of official methods for enforcement, and (4) the limitations and future prospects of the current methods.

Stable isotope dilution assay for the accurate determination of mycotoxins in maize by UHPLC-MS/MS

A fast, easy-to-handle and cost-effective analytical method for 11 mycotoxins currently regulated in maize and other cereal-based food products in Europe was developed and validated for maize. The method is based on two extraction steps using different acidified acetonitrile–water mixtures. Separation is achieved using ultrahigh-performance liquid chromatography (UHPLC) by a linear water–methanol gradient. After electrospray ionisation, tandem mass spectrometric detection is performed in dynamic multiple reaction monitoring mode. Since accurate mass spectrometric quantification is hampered by matrix effects, uniformly [13C]-labelled mycotoxins for each of the 11 compounds were added to the sample extracts prior to UHPLC-MS/MS analysis. Method performance parameters were obtained by spiking blank maize samples with mycotoxins before as well as after extraction on six levels in triplicates. The twofold extraction led to total recoveries of the extraction steps between 97% and 111% for all target analytes, including fumonisins. The [13C]-labelled internal standards efficiently compensated all matrix effects in electrospray ionisation, leading to apparent recoveries between 88% and 105% with reasonable additional costs. The relative standard deviations of the whole method were between 4% and 11% for all analytes. The trueness of the method was verified by the measurement of several maize test materials with well-characterized concentrations. In conclusion, the developed method is capable of determining all regulated mycotoxins in maize and presuming similar matrix effects and extraction recovery also in other cereal-based foods.

Determination of total sulfite in wine by ion chromatography after in-sample oxidation.

Sulfur dioxide (SO2) or sulfites are the most common preservatives used in winemaking. The level of total SO2 is subject to regulation. Currently, the regulatory determination of total SO2 (including sulfites) is done by the optimized Monier-Williams (OMW) method, which includes time-consuming distillation and titration steps. This paper describes the development and application of an alternative, rapid, straightforward, and reliable method for the determination of total sulfite in wine. In this method, a simple oxidation step using alkaline hydrogen peroxide (H2O2) solution is followed by ion chromatographic (IC) analysis of sulfate coupled with conductometric detection. Thirteen wines were analyzed in order to compare the in-sample oxidation method with the OMW-procedure. A t-test revealed satisfying compliance regarding sample preparation, i.e., alkaline H2O2 treatment and acidic distillation (OMW method). Comparable results were also obtained between IC analysis and acid/base titration. Our results indicate that the novel method (limit of quantification: 4 mg SO2 L(-1)) is well suited for the cost-efficient monitoring of regulatory limits.

Enantiomer-specific analysis of hexabromocyclododecane in fish from Etnefjorden (Norway).

High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was applied to sterospecifically quantify the content of alpha-, beta-, and gamma-hexabromocyclododecane (HBCD) in six fish species from the Norwegian Etnefjorden. A combination of a beta-PM cyclodextrin and an achiral column enabled the paired chromatographic separation of the stereoisomers in the order (-)-alpha-, (+)-alpha-, (-)-beta-, (+)-beta-, (+)-gamma- and, (-)-gamma-HBCD. The limits of detection were in the range of 6-21 pg g(-1) depending on the stereoisomer and the concentrations of alpha-, beta-, and gamma-HBCD in fillets ranged from <5.4 ng g(-1) to 11.1 microg g(-1) lipid weight. alpha-HBCD enantiomers were throughout dominating, and in most cases the accumulation of the respective first eluted enantiomers ((-)-alpha-, (-)-beta- and (+)-gamma-HBCD) was observed. Deviations from the racemic EF-value were considered to be significant if it was outside of the expanded uncertainty range for each of the racemic HBCD-ratios. The composition of HBCD isomers varied between the investigated fish species and the relative high values for the gamma-HBCD concentrations for the bottom-dwellers flounder and thorny skate seems to echo the HBCD pattern of ocean sediments.

Cocultivation of phytopathogenic Fusarium and Alternaria strains affects fungal growth and mycotoxin production

A laboratory study was conducted to evaluate the influence of cocultivation of toxigenic Fusarium (F.) and Alternaria (A.) fungi with respect to growth and mycotoxin production.

Photochemical trans-/cis-isomerization and quantitation of zearalenone in edible oils

The emphasis of the present work was to investigate the photochemical conversion of trans- to cis-zearalenone in edible oils under real-life conditions. For quantitation purposes a cis-zearalenone standard was synthesized and characterized for its identity and purity (≥95%) by (1)H NMR, X-ray crystallography, HPLC fluorescence and mass spectrometric detection. In a sample survey of 12 edible oils (9 corn oils, 3 hempseed oils) from local supermarkets all corn oils contained trans-zearalenone (median 194 μg/kg), but no cis-zearalenone was detected. For alteration studies trans-zearalenone contaminated corn oils were exposed to sunlight over 4 and 30 weeks, revealing an obvious shift toward cis-zearalenone up to a cis/trans ratio of 9:1 by storage in colorless glass bottles. Irradiation experiments of trans-zearalenone in different organic solvents confirmed the preferred formation of cis-zearalenone possibly caused by entropic effects rather than by enthalpic entities as investigated by quantum chemical and classical force field simulations.

Novel Solid-Phase Extraction for Epimer-Specific Quantitation of Ergot Alkaloids in Rye Flour and Wheat Germ Oil

Ergot alkaloids and their epimer-specific determination have gained increasing importance for food safety. A solid-phase extraction and cleanup method based on sodium-neutralized strong cation exchange (Na(+)-SCX) was developed to quantitate 12 priority ergot alkaloids in rye flour and wheat germ oil by HPLC fluorescence analysis. Sample preparation is achieved by omitting acidic and alkaline conditions enabling minimized epimerization, which is necessary to determine ergot alkaloids according to their natural distribution in foods. Ergot alkaloids are eluted from SCX-column by forming ion pairs using a sodium hexanesulfonate containing solution which prevents epimerization for at least 96 h. Method validation yielded recoveries of 80-120% (rye flour) and 71-96% (wheat germ oil) with a maximum limit of quantitation (LOQ) of 2.0 μg kg(-1) per ergot alkaloid for both matrices. The applicability of the developed method was demonstrated by analyzing 16 samples from German retail markets: 9 rye flours (max 178 ± 5 μg kg(-1)) and, reported for the first time, 7 wheat germ oils (max 56.8 ± 2.7 μg kg(-1)) expressed as the sum of 12 ergot alkaloids.

Mycotoxins as antagonistic or supporting agents in the interaction between phytopathogenic Fusarium and Alternaria fungi

The role of mycotoxins in the microbial competition in an ecosystem or on the same host plant is still unclear. Therefore, a laboratory study was conducted to evaluate the influence of mycotoxins on growth and mycotoxin production of Fusarium and Alternaria fungi. Fusarium culmorum Fc13, Fusarium graminearum Fg23 and two Alternaria tenuissima isolates (At18 and At220) were incubated on wheat kernels supplemented with alternariol (AOH), tetramic acid derivates (TeA), deoxynivalenol (DON) and zearalenone (ZEA) in an in vitro test system. Fungal biomass was quantified by determining ergosterol content. Three Fusarium toxins (DON, nivalenol and ZEA) and three Alternaria toxins (AOH, alternariol methyl ether (AME) and altenuene) were analysed by HPLC-MS/MS. If Alternaria strains grew in wheat kernels spiked with Fusarium mycotoxins, their growth rates were moderately increased, their AOH and AME production was enhanced and they were simultaneously capable of degrading the Fusarium mycotoxins DON and ZEA. In co...

Classical hybrid Monte-Carlo simulation of the interconversion of hexabromocyclododecane stereoisomers

The interconversion of the six main stereoisomers of the flame retardant hexabromocyclododecane (HBCD) is investigated by means of statistical thermodynamics using classical force-fields. ( ± )-α-, ( ± )-β- and ( ± )-γ-HBCD interconvert by swapping of absolute configurations on the three different (BrHC–CHBr)-moieties. The approach avoids saddle-point energy computations, but relies on classical thermodynamic simulation and pursues three consecutive steps. First, the application of classical hybrid Monte-Carlo simulations for quantum mechanical processes is justified. Second, the problem of insufficient convergence properties of hybrid Monte-Carlo methods for the generation of low temperature canonical ensembles is solved by an interpolation approach. Third, it is shown how free energy differences among stereoisomers are derived and how they can be used for the computation of interconversion rates. The simulation results confirm the experimentally observed interconversion rates and correctly identify α-HBCD as a thermodynamical sink in the oscillating mixture of stereoisomers.

(3S,11Z)-14,16-Dihy­droxy-3-methyl-3,4,5,6,9,10-hexa­hydro-1H-2-benz­oxacyclo­tetra­decine-1,7(8H)-dione (cis-zearalenone): a redetermination

The title compound, also known as cis-zearalenone (cis-ZEN), C18H22O5, has already been reported elsewhere [Griffin et al. (1981 ▶). ACA Ser. 29, 35], but no atomic coordinates are publicly available. The molecule is of interest with respect to its toxicity. In the crystal, intramolecular O—H⋯O hydrogen bonds stabilize the molecular conformation, while intermolecular O—H⋯O hydrogen bonds link the molecules to form infinite chains along the [110] and [1-10] directions. The absolute configuration has been assigned by reference to an unchanging chiral centre in the synthetic procedure.