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.

Type-B trichothecene calibrants: Comparison of HPLC and GC-results within an intercomparison study.

Within the EC-financed project "Feasibility Study for the Production of Certified Calibrants for the Determination of Deoxynivalenol and other B-Trichothecenes", an intercomparison study was performed with 13 European participants.Main goals of the intercomparison study were to check the feasibility of a small batch of gravimetrically prepared calibrants, to directly compare common and individually prepared calibrants, to test the practicability of toxin mixtures as calibrant solutions and finally to give recommendations for the means of certification. Additionally, it focused on the comparison of gas chromatography (GC) and high performance liquid chromatography (HPLC) for the determination of pure type-B trichothecene solutions, which is described in this publication.The participating laboratories received calibrant solutions as well as toxin solutions of unknown concentration and employed mainly HPLC-UV; GC-ECD (electron capture detection) and GC-MS (mass spectrometry) methods were used less often.The intercomparison study generally suffered from a high rate of outliers (22% of all the data). Throughout the study, 48% of all GC results were classified as outliers and it soon became apparent, that GC results highly infuenced the outcome of the study and that the used GC methods were not robust enough for the certification of type-B trichothecene calibrants. The high discrepancy between HPLC and GC results in the intercomparison study presumably lies in the crucial step of derivatisation.

Zearalenone in maize: stability testing and matrix characterisation of a certified reference material.

Within the certification process of a reference material for the determination of the mycotoxin zearalenone (ZON) in maize, short- and long-time stability tests of naturally contaminated maize have been performed. The short-term stability of ZON in the maize was evaluated under four different conditions (4, 25, 40 and 70 degrees C) in preliminary studies. Four storage times of 0, 1, 2 and 4 weeks were investigated using HPLC. The long-term stability study was conducted with measurements after 0, 3, 6, 12, 24 and 36 months under three storage conditions (4, 25 and 40 degrees C) in preliminary studies using HPLC. Stability data gained under two different conditions (4 and 25 degrees C) with five storage times of 0, 1, 6, 12 and 18 months were further evaluated for the contaminated maize in the certification process. Before the certification, the maize matrix had been characterized with respect to dry residue, ash content, fat content, protein content, ergosterol content and total dietary fibre, and the efficiency of gamma-irradiation on the fungal flora was investigated. The stability of the maize matrix was evaluated by monitoring UV absorption and ergosterol content under four different storage conditions (4, 25, 35 and 70 degrees C) with five storage times of 0, 1, 6, 12 and 24 months. Other possibly occurring mycotoxins (deoxynivalenol, nivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, fusarenon X and moniliformin) have been quantified. On the basis of the stability measurements, which showed no significant trends for both short- and long-term stabilities, it can be recommended to store the samples at temperatures < 4 degrees C and ship the samples at ambient temperatures.