Development and validation of a liquid chromatography/linear ion trap mass spectrometry method for the quantitative determination of deoxynivalenol-3-glucoside in processed cereal-derived products M. Suman ⇑ , E. Bergamini, D. Catellani, A. Manzitti Barilla Food Research Labs, via Mantova 166, 43100 Parma, Italy article info Article history: Available online 1 July 2012 Keywords: Deoxynivalenol-3-glucoside Masked mycotoxins Conjugated mycotoxins Food processing Bakery Bread Liquid chromatography Mass spectrometry Linear ion trap abstract Cereal-based food can be frequently contaminated by the presence of mycotoxins derived from Fusarium fungus, and, in particular, by deoxynivalenol (DON). Nowadays, analytical strategies for the detection of DON are well developed, but there are gaps for what concerns a correct identification, quantification and toxicological evaluation of the respective metabolites, mainly related to detoxifying actions via plant metabolism or to processing technologies and also referred to as ‘‘masked’’ mycotoxins. Here, we report the development of a liquid chromatography/linear ion trap mass spectrometry method capable of determining deoxynivalenol-3-glucoside (DON-3G), which is the main known DON metabolite, in different processed cereal-derived products. Samples were extracted with a mixture of methanol/water (80:20; v/v) and cleaned up using immunoaffinity columns. Chromatographic separation was performed using a core–shell C 18 column with an aqueous acetic acid/methanol mixture as the mobile phase under gradient conditions. The method was in-house validated on a bread matrix as fol- lows: matrix-matched linearity (r 2 > 0.99) was established in the range of 10–200 lg kg 1 ; trueness expressed as recovery was close to 90%; good intermediate precision (overall RSD < 9%) and adequate detection quantitation limits (4 and 11 lg kg 1 , respectively) were achieved. Furthermore, applying a metrology approach based on intralaboratory data, the estimated measurement expanded uncertainty was determined to be equal to 29%. The reliability of the method was finally demonstrated in bread, cracker, biscuit and minicake commodities, resulting in relatively low levels of DON-3G, which were not higher than 30 lg kg 1 . Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Mycotoxins are low molecular compounds, produced by the secondary metabolism of fungi able to attack a wide group of cere- als, such as wheat, maize, barley, oat and rye. They can exert toxic and/or carcinogenetic actions in animals and humans, in some cases even at low concentrations close to lg kg 1 levels, (Bennet & Klich, 2003; Hussein & Brasel, 2001; Sudakin, 2003) in food and feed. Weather conditions, agricultural practices, and other environmental and contingent factors can strongly influence their incidence in food crops from year to year. Deoxynivalenol (DON) is the most important toxin for wheat and wheat-derived products due to its worldwide occurrence and potential toxicological impact on animal and human health through immunomodulation, protein synthesis inhibition, ribo- some disaggregation, mitochondrial function compromise, lipid peroxidation, and cellular death (Prelusky, Rotter, & Rotter 1994). Mycotoxins can be covalently or non-covalently bound to phen- olics, proteins, cell wall polysaccharides, lipids and lignin (Lamou- rex & Rusness, 1986). Cereal crops exposed to deoxynivalenol (DON) infection are capable of detoxifying this mycotoxin through plant metabolism. One of the most important resistance mecha- nisms is the detoxification of DON into 3-ß-D-glucopyranosyl be- fore compartmentalisation of this metabolite into vacuoles or cell walls (Poppenberger et al., 2003). Approximately 600 million tons of wheat is produced per year worldwide, and most of the wheat is converted to wheat flour for human consumption and processed into various foods, such as breads, pastas, noodles, and cakes (Pacin, Ciancio Bovier, Cano, Taglieri, & Hernandez Pezzani, 2010). Fusarium toxins are generally quite stable during commercial food processing (Masayo, 2008) and cannot be completely elimi- nated, which leads to potential contamination of finished cereal- based foods. To protect consumers from an unacceptably high dietary intake of trichothecenes, maximum levels have been set in EU legislation (European Commission, 2006). For example, deoxynivalenol 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.06.085 ⇑ Corresponding author. Address: Barilla G.R.F. lli SpA – Food Research Labs, via Mantova 166, 43100 Parma, Italy. Tel.: +39 0521 262332; fax: +39 0521 263452. E-mail address: michele.suman@barilla.com (M. Suman). Food Chemistry 136 (2013) 1568–1576 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem