Simultaneous determination of Deoxynivalenol, Deoxynivalenol-3-Glucoside and Nivalenol in wheat grains by HPLC-PDA with immunoaffinity column cleanup

Deoxynivalenol-3-glucoside (D3G) is a modified mycotoxin formed by the metabolism of plants through the conjugation of deoxynivalenol (DON) with glucose. Toxicology studies of D3G for human and animal health are still under investigation, and the development of practical and reliable methods for its direct determination, especially in cereal matrices, is of great importance. In the present study, a methodology for simultaneous determination of D3G, DON, and nivalenol (NIV) in wheat grains, using immunoaffinity column (IAC) cleanup, separation by C18 column and detection by ultraviolet (UV) absorption, was optimized and in-house validated. The results demonstrated adequate values of D3G recovery from IAC and spiked samples. Intraday precision, linearity, limit of detection and limit of quantification (LOQ) were also adequate for the determination of these mycotoxins. Range of applicability varied from 47.1 to 1000 g/kg for D3G and from 31.3 to 1000 g/kg for DON and NIV, with recovery ranging from 84.7±7.2 % to 112.3±8.1Felipe Trombete is grateful for a doctoral fellowship provided by the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES)

Determining mycotoxins and mycotoxigenic fungi in food and feed

Over the last few decades it has become increasingly clear that mycotoxins play a significant role in food and feed safety. Indeed, mycotoxins have been shown to be the principal threat regarding chronic toxicity. Legislative limits for a range of mycotoxins continue to develop worldwide resulting in an increased number of official controls deriving from national food safety plans and for food trade purposes. This book therefore focuses on recent developments in the determination of mycotoxins and mycotoxigenic fungi in food and feed. A mycotoxin test procedure is a multi-stage process generally consisting of three steps: sampling, sample preparation and analytical determination. The sampling phase is the largest source of variability of the test procedure. The official sampling protocols are still complicated and very challenging in practical terms. Further extensive research on sampling plans is mandatory, taking into account the real risk to human health together with the economic perspective. New developments in sample preparation focus on faster, environmentally friendly, cost effective and fit-for-purpose extraction and clean-up methods in food, feed, biological tissue and bodily fluids. Screening immunochemical and confirmatory chromatographic analytical methods are widely used; a clear trend towards multimycotoxin analysis and more precisely towards LC–MS/MS has been noticed. Quality assurance in mycotoxin analysis is of the utmost importance. Notwithstanding the general acceptance of the benefits of adopting a performance criteria-based approach, some countries have a regulatory framework which requires the publication of ‘official methods’ in their own regulations. Food control laboratories should continuously follow actual progress in analysis development and statistical method validation within an accredited quality environment such as prescribed in the ISO 17025 norm. Further attention towards a harmonized method validation procedure is necessary. In order to understand possible links between mycotoxins and human disease or animal disease outbreaks, it is necessary to measure the exposure to the toxin in question. Advances in analytical techniques have resulted in the development and use of various biological markers (biomarkers) which allow more accurate and objective assessment of exposure at the individual level. The development and determination of validated exposure as well as mechanism-based biomarkers is critical to reduce the existing uncertainty in the risk assessment of most mycotoxins. Fungal isolates involved in mycotoxicoses are preferably identified by a polyphasic approach in order to avoid mistakes, starting at genus level and further to species level using a combination of morphological, physiological, nutritional and chemical data. The identification is validated by PCR-based molecular methods which can be considered under two main complementary approaches: by targeting conserved functional genes or regions of taxonomical interest, or by focusing on the mycotoxigenic genes. The possibility of using a highly standardized, rapid and practical DNA barcoding protocol that can be easily used both by researchers involved in species definition studies and by non-experts for practical uses is currently investigated. However, in order to assess the risks related to the presence of mycotoxigenic fungi in food and feedstuffs reliably, one should also investigate whether or not the mycotoxin genes are expressed. Further progress in transcriptomics, proteomics and metabolomics will continue to advance the understanding of fungal secondary metabolism, providing insight into how to reduce mycotoxin contamination of crop plants and the food/feed derived therefrom. Fungal secondary metabolites, mycotoxins and food safety will continue to be of critical interest to a variety of researchers for years to come. Innovations take place at a rapid pace, for example through new nanotechnology-based biosensing techniques and non-destructive spectroscopic techniques. Furthermore, the discovery of masked mycotoxins and the inherent analytical challenges will be the subject of future research