Chemical Synthesis of Deoxynivalenol-3-β-d-[13C6]-glucoside and Application in Stable Isotope Dilution Assays

Abstract

Abstract:Modified mycotoxins have been gaining importance in recent years and present a certain challenge in LC-MS/MS analysis. Due to the previous lack of a labeled isotopologue of the modified mycotoxin deoxynivalenol-3-glucoside, in our study we synthesized the first 13 C-labeled internal standard. Therefore, we used the Königs-Knorr method to synthesize deoxynivalenol-3-β-D-[ 13 C 6 ]-glucoside originated from unlabeled deoxynivalenol and [ 13 C 6 ]-labeled glucose. Using the synthesized isotopically-labeled standard deoxynivalenol-3-β-D-[ 13 C 6 ]-glucoside and the purchased labeled standard [ 13 C 15 ]-deoxynivalenol, a stable isotope dilution LC-MS/MS method was firstly developed for deoxynivalenol-3-glucoside and deoxynivalenol in beer. The preparation and purification of beer samples was based on a solid phase extraction. The validation data of the newly developed method gave satisfying results. Intra-and interday precision studies revealed relative standard deviations below 0.5% and 7%, respectively. The recoveries ranged for both analytes between 97% and 112%. The stable isotope dilution assay was applied to various beer samples from four different countries. In summary, deoxynivalenol-3-glucoside and deoxynivalenol mostly appeared together in varying molar ratios but were quantified in rather low contents in the investigated beers.

Recent Advances and Future Challenges in Modified Mycotoxin Analysis: Why HRMS Has Become a Key Instrument in Food Contaminant Research

“…A possible approach to cope with the matrix effect is represented by stable isotope dilution assay (SIDA) [26]. Multi-mycotoxins methods applying this technique have been developed [27,28], also thanks to in-house synthesis of labeled isotopologue mycotoxin standards, including isotopologues of modified forms (i.e., DON3Glc, 3Ac-DON, 15Ac-DON) [29]. Authenticity and method performance have been demonstrated by analyzing naturally contaminated samples, such as malt, beer and maize [30].…”

Synthesis of Isotope‐Labeled Deoxynivalenol‐15‐O‐Glycosides

“…For the development of improved and accurate analytical methods and toxicological risk assessment, sufficient quantities of these compounds are required in high purity. Furthermore, isotope‐labeled conjugates are crucial for accurate quantification of mycotoxin metabolites by LC–MS , …”

“…The synthesis of 13 C‐labeled DON‐3‐ O ‐β‐ d ‐[ 13 C 6 ]glucoside was accomplished by Königs–Knorr glycosylation (Ag 2 CO 3 in dichloromethane) by Habler et al very recently. However, the authors reported several drawbacks of their method: (i) an excess of isotope‐labeled glucosyl donor; (ii) a very long reaction time (5 d); and (iii) failed scale‐up of the reaction …”

“…[6] For the development of improved and accurate analytical methods and toxicological risk assessment, sufficient quantities of these compounds are required in high purity. Furthermore, isotope-labeled conjugates are crucial for accurate quantification of mycotoxin metabolites by LC-MS. [6,10] Previously described glycosylation reactions of DON were carried out under Königs-Knorr conditions (CdCO 3 in toluene under reflux) in 38 % yield. [11] Recent advances have been made in the preparation of DON-3-O--D-glucoside by an enzymatic approach.…”

“…However, the authors reported several drawbacks of their method: (i) an excess of isotope-labeled glucosyl donor; (ii) a very long reaction time (5 d); and (iii) failed scale-up of the reaction. [10] In this paper, we present the development of an efficient general procedure for the preparation of DON-15-O-glycosides, and the synthesis of isotope-labeled analogs using a 13 C 6labeled N-phenyltrifluoroacetimidate glucosyl donor.…”