Critical evaluation of indirect methods for the determination of deoxynivalenol and its conjugated forms in cereals

Abstract

A critical assessment of three previously published indirect methods based on acidic hydrolysis using superacids for the determination of “free” and “total” deoxynivalenol (DON) was carried out. The modified mycotoxins DON-3-glucoside (D3G), 3-acetyl-DON (3ADON), and 15-acetyl-DON (15ADON) were chosen as model analytes. The initial experiments focused on the stability/degradation of DON under hydrolytic conditions and the ability to release DON from the modified forms. Acidic conditions that were capable of cleaving D3G, 3ADON, and 15ADON to DON were not found, raising doubts over the efficacy of previously published indirect methods for total DON determination. Validation of these indirect methods for wheat, maize, and barley using UHPLC-MS/MS was performed in order to test the accuracy of the generated results. Validation data for DON, D3G, 3ADON, and 15ADON in nonhydrolyzed and hydrolyzed matrices were obtained. Under the tested conditions, DON was not released from D3G, 3ADON, or 15ADON after hydrolysis and thus none of the published methods were able to cleave the modified forms of DON. In addition to acids, alkaline hydrolysis with KOH for an extended time and at elevated temperatures was also tested. 3ADON and 15ADON were cleaved under the alkaline pH caused by the addition of KOH or aqueous K2CO3 to “neutralize” the acidic sample extracts in the published studies. The published additional DON increase after hydrolysis may have been caused by huge differences in matrix effects and the recovery of DON in nonhydrolyzed and hydrolyzed matrices as well as by the alkaline cleavage of 3ADON or 15ADON after the neutralization of hydrolyzed extracts.

Simultaneous Determination of Deoxynivalenol, Deoxynivalenol-3-Glucoside and Nivalenol in Wheat Grains by HPLC-PDA with Immunoaffinity Column Cleanup

“…The hydrolysis procedure was considered by these authors as a cheaper and simpler alternative to determine total DON-conjugated mycotoxins in cereals, especially because it uses quantification by ELISA kits. However, Malachová et al (2015) recently published a critical evaluation of these indirect methods for total DON-conjugated determination, raising doubts on their efficacy as, according to these authors, none of them were able to release DON from its conjugated form, and for this reason, they were not recommended for such evaluations.…”

Biochemical Characterization of a Recombinant UDP-glucosyltransferase from Rice and Enzymatic Production of Deoxynivalenol-3-O-β-D-glucoside

“…It was shown that the bioavailability of D3G is probably low compared to that of DON, as human Caco-2 cells do not absorb D3G [ 33 ]. Although D3G is highly resistant to acidic hydrolysis [ 34 ], it can be enzymatically hydrolyzed by intestinal bacteria [ 35 ] and the released DON may be (partially) absorbed in the distal part of the gut. It was shown that D3G is indeed effectively hydrolyzed in the digestive tracts of humans and animals [ 36 , 37 , 38 , 39 ].…”

Hydrolysers of modified mycotoxins in maize: α-Amylase and cellulase induce an underestimation of the total aflatoxin content

“…Some authors pointed out that hidden fumonisins could be embedded to starch or proteins of the matrix. Respecting DON, alkaline conditions cause a transformation of 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) to DON (Malachova, Stockova, Wakker, Varga, Krska, Michlmayr, et al, 2015). These authors showed that 32 % of 3-ADON and 47 % of 15-ADON in flour matrix were hydrolysed to DON when samples were submitted to KOH treatment.…”

“…These authors showed that 32 % of 3-ADON and 47 % of 15-ADON in flour matrix were hydrolysed to DON when samples were submitted to KOH treatment. This was caused by the presence of additional (unknown) sources of DON in the sample (Malachova, et al, 2015). Some DON could be hidden in starch or proteins of the matrix like fumonisins.…”