Feasibility study for the production of certified calibrants for the determination of deoxynivalenol and other B-trichothecenes: intercomparison study.

Thirteen European laboratories experienced in the analysis of mycotoxins participated in an intercomparison study within a European Commission-funded project. Goals of the study were to check the fitness for purpose of a small batch of gravimetrically prepared calibrants; to compare individually prepared calibrants with common calibrants; to check the feasibility of toxin mixtures as calibrant solutions; and to give recommendations on the production of future certified reference materials (CRMs) with regard to the nature of the calibrant and the means of certification. Each laboratory received ampules of each common calibrant containing single toxins [solution containing either deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), nivalenol (NIV), or 15-acetyl-DON (15-Ac-DON)] and 3 ampules of toxin-mixture (solutions of DON + 3-Ac-DON + NIV in acetonitrile) of known concentrations (about 20 microg/mL). Ampules with single toxins (solution containing either DON, 3-Ac-DON, NIV, or 15-Ac-DON) and a toxin-mixture (solutions of DON + 3-Ac-DON + NIV in acetonitrile) of unknown concentrations were distributed to the participants for quantification. The participating laboratories used mainly high-performance liquid chromatography (HPLC)-diode array detection UV for DON, 3-Ac-DON, NIV, and 15-Ac-DON; gas chromatography-electron capture detection (GC-ECD) and GC-mass spectrometry methods were used sparingly. Linear calibration curves were achieved by >90% of the participants. Relative between-day variation (RSDr) of 26% of the laboratories was greater than the target value of 5% for HPLC, and RSDr of 32% of the laboratories was greater than the desired value of 10% for GC. Relative between-laboratory variation (RSDR) of the GC results obtained with single common calibrants was greater than the target value of 16% for all laboratories. RSDR of the HPLC results for the common unknown single toxin solutions was less than the target value of 8% except for 15-Ac-DON. Generally, better recoveries were observed from common calibrants (102% for mix calibrants and 98% for single calibrants) than from individually prepared calibrants (95%). This international comparison study clearly showed the high scattering of results in the analysis of type-B trichothecenes, particularly when GC was used. Obviously, this intercomparison study was not suited for the certification of B-trichothecenes. A certification of the proposed calibrant material was therefore recommended on the basis of its gravimetrical preparation.

Advances in the analysis of mycotoxins and its quality assurance.

This article covers the latest activities in mycotoxin analysis and the advances of its respective quality assurance. The majority of mycotoxin analyses carried out in the laboratories is still based on physicochemical methods, which are continually improved. For example, immunoaffinity columns and multifunctional clean-up columns have become of increasing importance and in some areas of mycotoxin analysis they have more or less displaced conventional liquid-liquid partitioning or column chromatography during clean-up. The need for rapid yes/no decisions on the other hand has led to a number of new screening methods. In particular, rapid and easy-to-use test kits based on immunoanalytical principles or the generation of artificial macromolecular receptors employed in molecularly imprinted polymers (MIPs) have made good progress. Further research in mycotoxin analysis is pursued in the field of biosensors and also the potential of infrared spectroscopic techniques as screening method has been demonstrated. In the area of multi mycotoxin analysis the most promising development was observed in mass spectrometry. At the same time, several interlaboratory studies in the field of mycotoxin analysis revealed problems proven by high between laboratory standard deviation and non-traceable results. This not only shows the necessity of reliable methods and well defined performance characteristics but also the need for appropriate calibrants of defined concentration and stated purity. A certified zearalenone (ZON) calibrant is already available and a certified calibrant containing various trichothecenes is currently under development. (Certified) reference materials are available for aflatoxins in a number of commodities, ochratoxin A (OTA) in wheat, deoxynivalenol (DON) in maize and wheat, and ZON in maize. With these measures important steps towards traceability of results in mycotoxin analysis have been achieved.

Purity assessment of crystalline zearalenone.

Commercially available solid zearalenone (ZON) to be used as a certified liquid calibrant (BCR-699) in a project funded by the European Commission within the Standard Measurement and Testing program was characterized and its purity determined. The degree of purity of the ZON was examined by UV spectrophotometer, liquid chromatography (LC) with diode array and fluorescence detection, 1H and 13C-NMR spectrometry, LC-mass spectrometry (LC/MS/MS), ion chromatography (IC), and differential scanning calorimetry (DSC). The diagrams obtained from DSC analysis and the UV spectrum showed no detectable impurities. Likewise, no impurities were observed by LC analysis with both diode array and fluorescence detection. IC determination revealed negligible contamination of ZON with chloride of 0.020 +/- 0.005% and nitrate of 0.016 +/- 0.006%. Zearalanone (ZAN) was identified as one of 2 minor (0.2%) impurities by LC/MS/MS. The 1H-NMR measurements revealed an additional peak, which has not been previously reported in the literature. It could be identified as part of the ZON spectrum as the signal arising from the phenolic proton attached to C4'. The manufacturer states an additional contamination with 0.2% methylene chloride, which could be confirmed to an extent of 0.1% by 1H-NMR. Minor impurities, whose structures remain unknown, were discovered at 3.5 and < 1 ppm. Total percentage of impurities based on NMR measurement was estimated not to exceed 1%. A purity of 99.5% with a tolerance of +/- 0.5% was finally attributed to the ZON studied in this project.