Malachová Alexandra, Štočková Lenka, Wakker Astrid, Varga Elisabeth, Krska Rudolf, Michlmayr Herbert, Adam Gerhard, Berthiller Franz
Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 20, 3430, Tulln, Austria,
Anal Bioanal Chem. 2015 Aug;407(20):6009-20. doi: 10.1007/s00216-015-8793-0. Epub 2015 Jun 12.
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.
对三种先前发表的基于使用超强酸进行酸性水解来测定“游离”和“总”脱氧雪腐镰刀菌烯醇（DON）的间接方法进行了批判性评估。选择了修饰的霉菌毒素DON - 3 - 葡萄糖苷（D3G）、3 - 乙酰基 - DON（3ADON）和15 - 乙酰基 - DON（15ADON）作为模型分析物。初始实验聚焦于DON在水解条件下的稳定性/降解以及从修饰形式中释放DON的能力。未发现能够将D3G、3ADON和15ADON裂解为DON的酸性条件，这引发了对先前发表的用于测定总DON的间接方法有效性的质疑。使用超高效液相色谱 - 串联质谱法（UHPLC - MS/MS）对这些间接方法在小麦、玉米和大麦中的应用进行了验证，以测试所得结果的准确性。获得了非水解和水解基质中DON、D3G、3ADON和15ADON的验证数据。在测试条件下，水解后DON未从D3G、3ADON或15ADON中释放出来，因此所发表的方法均无法裂解DON的修饰形式。除了酸之外，还测试了用氢氧化钾在高温下长时间进行碱性水解。在已发表的研究中，通过添加氢氧化钾或碳酸钾水溶液以“中和”酸性样品提取物所导致的碱性pH条件下，3ADON和15ADON被裂解。水解后所发表的额外DON增加可能是由于基质效应的巨大差异以及非水解和水解基质中DON的回收率差异，以及水解提取物中和后3ADON或15ADON的碱性裂解所致。
