Certified reference materials and proficiency testing.
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Journal Article↗
Biomedical subjects
R Krska
Publications and source records attributed to R Krska.
Improving methods of analysis for mycotoxins: molecularly imprinted polymers for deoxynivalenol and zearalenone.
Selective polymeric phases intended for future use in separation/extraction of deoxynivalenol and zearalenone from beverages have been prepared. Using crystalline deoxynivalenol, zearalenone and quercetin, molecularly imprinted polymers were obtained by a non-covalent imprinting approach via a photo-initiated addition polymerization. Prepared polymers were based on 4-vinylpyridine, methacrylic acid or 2-trifluoromethylacrylic acid as the functional monomer and on ethyleneglycol dimethacrylate, trimethyltrimethacrylate or divinylbenzene as the cross-linking monomer. Selectivity of the generated molecularly imprinted polymers has been investigated by application of the prepared molecularly imprinted polymers as stationary phases in high-pressure liquid chromatography experiments. The retention and elution behaviours of the template compounds and structurally related substances were determined and compared. The results promise future application of molecularly imprinted polymers as alternative selective matrices for clean-up and enrichment of deoxynivalenol and zearalenone.
Beer↗
Determination of moniliformin in maize by ion chromatography.
An ion chromatographic method for the determination of the mycotoxin moniliformin in maize was developed. The method contains a fast and simple clean-up procedure, which allows high volume sample injection. No further pre-concentration step is required. The limit of quantification was estimated to be 0.12 mg/kg moniliformin in maize. In contrast to the two previously published ion chromatographic methods, the moniliformin peak is base line separated and occurs in a region of a smooth base line in the ion chromatogram. The newly developed method showed a recovery through the whole analytical procedure of 97+/-3.5 %, whereas most previously described analytical methods had recoveries in the 70% range.
Chromatography, High Pressure Liquid↗
Structural characterization of metabolites after the microbial degradation of type A trichothecenes by the bacterial strain BBSH 797.
Contamination of feed with trichothecenes, a group of Fusarium mycotoxins, leads to losses in performance due to their immunosupressive effects and the negative effect on the gastrointestinal system in animal production. A possible way of detoxification is microbial degradation, which was the focus of this study. A bacterial strain--BBSH 797--which can degrade some mycotoxins of the trichothecene group, has already been isolated. It transforms deoxynivalenol (DON) into its metabolite DOM-1, the non-toxic deepoxide of DON. Analogous to the microbial degradation of DON, the transformation of six different type A trichothecenes was observed. The metabolites appearing were characterized by GC-MS after derivatization with TRI-SIL TBT. Two metabolites were additionally, identified by liquid chromatography-mass spectrometry with particle beam interface (LC-PB-MS) with electron impact (EI)-ionization mode. The major finding was that scirpentriol was completely transformed into its non-toxic metabolite deepoxy scirpentriol, while the mycotoxin T-2 triol underwent a more complicated metabolism. According to the study, T-2-triol was degraded into its non-toxic deepoxy form and into T-2 tetraol, which was then further metabolized to deepoxy T-2 tetraol. GC-MS after derivatization with TRI-SIL TBT was suitable for the structural characterization of trichothecenes and their degradation products. Besides the mass spectra of already known degradation products, spectra of new metabolites could be recorded by LC-PB-MS.
Animals↗
The state-of-the-art in the analysis of estrogenic mycotoxins in cereals.
The increasing public awareness of chemicals that mimic or otherwise interfere with the activity of natural hormones - so-called endocrine disrupters - has also led to greater study of mycotoxins with estrogenic potential. The purpose of this paper is to introduce the topic of estrogenic mycotoxins and to discuss the state-of-the-art in the analysis of these substances in cereals, with special emphasis on zearalenone (ZON) as its most relevant representative. Because the use of immunoaffinity columns (IAC) followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and immunoassays are currently the most frequently used methods for the determination of ZON and its metabolites, these techniques are discussed in more detail. Recent papers, which have revealed the great potential of HPLC-MS(MS) for the simultaneous detection and identification of several estrogenic mycotoxins, are discussed. The performances of the state-of-the-art methods are finally compared by study of the results obtained in recent international intercomparison studies. On the one hand, these studies revealed the good performance of both chromatographic and antibody-based methods. On the other hand, the need for better means of external quality assurance measures, especially the availability of certified reference materials and certified standards, has clearly been demonstrated.
Chromatography, High Pressure Liquid↗
The state-of-the-art in the analysis of type-A and -B trichothecene mycotoxins in cereals.
The aim of this review is to describe the state-of-the-art in the analysis of A- and B-trichothecene mycotoxins in cereals and to support knowledge and experience exchange between laboratories in the field of Fusarium mycotoxin analysis. Current screening tests and quantitative methods for the most prevalent type-A and -B trichothecenes, HT-2 and T-2-toxin, and deoxynivalenol (DON) are reviewed. This includes the extraction and clean-up procedures and chromatographic methods (TLC, HPLC, GC) applied and the immunochemical methods, especially enzyme-linked immunosorbent assay (ELISA), employed for the determination of these mycotoxins. Results from recent intercomparison studies of the determination of DON are also discussed. Experience gained during these intercomparisons clearly shows the need for further improvement in the determination of trichothecenes, to obtain more accurate and comparable results. This also indicates there is a strong need for the development of further certified reference materials (CRM) which would enable comparison of measurement results between different European laboratories for several A- and B-trichothecenes. For both A- and B-trichothecenes there is still a lack of simple and reliable screening methods enabling the rapid detection of these mycotoxins at low cost.
Chemistry Techniques, Analytical↗
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.
Calibration↗
An enzyme linked immunoassay for the determination of deoxynivalenol in wheat based on chicken egg yolk antibodies.
An indirect competitive enzyme linked immunoassay (ELISA) for the detection of the Fusarium mycotoxin deoxynivalenol (DON) in wheat was developed. Instead of the much more common antibody isolation from mammal serum, DON specific antibodies were, for the first time, isolated from the eggs of previously immunized hens. The limit of detection was 2 microg/L for standard curves and spiked wheat extracts. Recoveries for naturally contaminated samples (200-525 microg/kg) were between 80 and 125% compared with GC-ECD data. Concentrations for naturally contaminated samples were chosen with regard to current Austrian guidelines concerning DON levels in produce intended for human consumption, recommending a maximum of 500 microg DON/kg.
Animals↗
The effect of the Fusarium metabolite beauvericin on electromechanical and -physiological properties in isolated smooth and heart muscle preparations of guinea pigs.
The electromechanical and -physiological effects of beauvericin were studied in isolated smooth and heart muscle preparations of the guinea pig. Beauvericin concentration-dependently decreased the force of contraction in precontracted (60 mM KCl) terminal ilea with an IC50 of 0.86 microM, and in electrically stimulated (1 Hz) papillary muscles with an IC50 of 18 microM. This negative inotropic effect in papillary muscles was antagonised in a non-competitive way by increased extracellular calcium concentrations. Spontaneous activity in right atria was affected at concentrations > 10 microM beauvericin. The negative chronotropic effect was less pronounced than the negative inotropic effect. In action potentials of electrically driven (1 Hz) papillary muscles, 10 microM beauvericin significantly decreased membrane resting potential until unexcitability of the preparation occurred. Despite depolarisation of the membrane the maximum rate of rise of the action potential was not changed. The action potential duration was shortened, but the decrease was only significant at times to 20% and 50% repolarisation. These data, derived from the electrophysiological experiments, not only imply an effect on the calcium current as suggested by the effects on contractility, but also an interaction with the sodium inward and potassium outward currents.
Action Potentials↗
High-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry as a new tool for the determination of the mycotoxin zearalenone in food and feed.
A new method for the determination of the mycotoxin zearalenone (ZON) in food and feed, based on HPLC-MS with an atmospheric-pressure chemical ionization (APCI) interface after extraction from cereals and clean-up by either conventional solid-phase or immunoaffinity cartridge is presented. The APCI interface parameters are optimized to provide detection of ZON with maximum sensitivity after RP separation of ZON on a C18 column with acetonitrile-water (40:60, v/v) at 1 ml/min column flow without split. Using APCI-MS detection, the sensitivity of the method was improved by a factor of ca. 50 in comparison to HPLC with fluorescence detection, allowing determination of ZON down to 0.12 microgram/kg maize which is well below present threshold values. Due to the selectivity of MS detection, it also was possible to quantitatively determine ZON both in raw extracts without clean-up using a normal-size (100 mm) chromatographic column or using only a short (20 mm) chromatographic column, when a clean-up was done to minimize possible interferences.
Animal Feed↗
Performance of modern sample preparation techniques in the analysis of Fusarium mycotoxins in cereals.
The efficiency of modern sample preparation techniques are discussed and compared to well-established techniques with respect to the determination of zearalenone in corn and B-trichothecenes in wheat in the microgram/kg range. This includes the use of immuno-affinity columns and of multifunctional Mycosep columns as well as the employment of supercritical fluid extraction for the trace analysis of these major Fusarium mycotoxins. In addition, the performance of new analytical methods was investigated in an interlaboratory comparison study only recently organized by our laboratory. From both the validation data, and from the results of the intercomparison study, the suitability and competitiveness of the described methods could be clearly demonstrated.
Chromatography, Affinity↗
Determination of trichothecene mycotoxins in wheat by use of supercritical fluid extraction and high-performance liquid chromatography with diode array detection or gas chromatography with electron capture detection.
The extraction behaviour of the Fusarium mycotoxin deoxynivalenol (DON) and some related type B trichothecenes from spiked seasand, spiked wheat flour and naturally contaminated wheat flour with modified supercritical CO2 has been investigated and optimized under several conditions. The extraction fluid was decompressed over a solid-phase trap and the amount of deposited analytes was determined by HPLC-diode array detection (DAD) or GC-electron capture detection (ECD) without any further clean-up. Recovery rates as high as 90.1 +/- 10.7% were achieved for spiked wheat samples and 53.0 +/- 3.2% for naturally contaminated samples. The performance of the optimized supercritical fluid extraction (SFE) method was compared with an already well established analytical method employing extraction on a rotary shaker in combination with Mycosep clean-up. Moreover, the SFE procedure developed for naturally DON contaminated wheat was employed for the simultaneous extraction of 5 type B trichothecenes by GC-ECD. This work represents the first successful approach in obtaining an SFE-method for the extraction of Fusarium mycotoxins from wheat with reasonable recoveries and good precision.
Chromatography, Gas↗
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.
Anti-Bacterial Agents↗