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

Abstract

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.

A Modified Approach to Evaluation of DON Content in Scab-Damaged Ground Wheat by Use of Diffuse Reflectance Spectroscopy

“…Only rarely was ergosterol content reported (Muthomi et al 2002) at the level of some hundreds parts per million while typically it is by a factor of one or two ranges of magnitude lower (Seitz et al 1977;Wiśniewska and Buśko 2005;Kos et al 2003Kos et al , 2004. The well-known DON and other trichothecenes absorption band at 218-220 nm (Krska et al 2005) certainly influences observed UV spectra; however, DON concentration at the parts per million range cannot be used for direct quantification of this substance from diffuse reflectance spectra because of not only low concentration but also because of the overlapping of DON band with proteins bands. No likely assignment has been found for a weaker peak in the plot of variance seen at 380 nm, though it could be deduced from the measurements reported by Birlouez-Aragon et al (2004) that an absorption band of lysine can appear at this wavelength.…”

Within-field variability of Fusarium head blight pathogens and their associated mycotoxins

“…Clean-up columns used to pre-purify samples were purchased from Rhone Diagnostic Technologies Ltd. (UK). Trichothecene standard stock and working solutions were prepared according to Krska et al (2005). The vials were sealed and stored in the dark at 4°C; fresh working solution was prepared each week.…”

“…Trichothecenes were extracted according to Krska et al (2005) using 10 g of sample and 50 ml of an acetonitrile-methanol as extraction mixture (84:16, v/v). The final solution was filtered with cellulose filter through a 0.22 μl syringe filter (Millipore, Bedford, MA, USA) prior to injection (20 μl) onto the LC MS.…”

Stable isotope dilution analysis of the Fusarium mycotoxins deoxynivalenol and 3‐acetyldeoxynivalenol

“…Compound 9 is the oxidation product of 3-acetyl-7-deoxy-DON (8, Scheme 2), which was an impurity in our 3-AcDON. The hydrolysis product of 8 (7-deoxy-DON) is furthermore described to be a major impurity even of commercially available DON [24].…”