Abstracts (WP5)

Simultaneous LC–MS/MS determination of aflatoxin M1, ochratoxin A, deoxynivalenol, de-epoxydeoxynivalenol, α and β-zearalenols and fumonisin B1 in urine as a multi-biomarker method to assess exposure to mycotoxins

Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M1 (AFM1), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alphazearalenol (α-ZOL)/beta-zearalenol (β-ZOL), and fumonisin B1 (FB1) can be used as urinary biomarkers. A liquid chromatographic–tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column.

Separation of the biomarkers was performed by reversed phase chromatography using a multi-step linear methanol– water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC– ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03–12 ng mL−1, using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3–20%. Enzymatic digestion with β- glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time.

Deoxynivalenol impairs porcine intestinal barrier function and decreases the protein expression of claudin-4 through a Mitogen Activated Protein kinase dependant mechanism.

Deoxynivalenol (DON) is a common mycotoxin that contaminates cereals and their by-products. The gastrointestinal tract is the first physical barrier against ingested food contaminants. DON contributes to the loss of barrier function of the intestine through the decreased expression of claudin-4 protein, a tight junction protein. The mechanism by which DON alters the intestinal barrier function remains poorly characterized. Therefore, we investigated the involvement of mitogen-activated protein kinases (MAPK) in the DON-induced loss of barrier function. We first verified that 30 μmol/L of DON activated MAPK in a highly sensitive porcine intestinal epithelial cell line (IPEC-1). Inhibition of p44/42 extracellular signal-regulated kinase (ERK) phosphorylation, with 0.5 μmol/L of the specific MAPK pharmacological inhibitor U0126 for 2 h, restored the barrier function of the differentiated intestinal epithelial cell monolayers. The restoration of barrier function was evaluated by trans-epithelial electrical resistance measurements and tracer flux paracellular permeability experiments. The U0126 also restored the intestinal expression of claudin-4 protein, thereby demonstrating that MAPK activation is involved in claudin-4 protein expression and claudin-4 is involved in the maintenance of the intestinal epithelial cell barrier function. Further experiments indicated that p44/42 ERK is not involved in the transcriptional regulation of claudin-4. In conclusion, we demonstrated that DON-induced activation of the p44/42 ERK signaling pathway inhibits the expression of claudin-4 protein, which leads to impaired intestinal barrier function. Given the high levels of DON in cereal grains, these observations of impaired barrier function have implications for human and animal health.

Comparison of methods and optimisation of the analysis of fumonisins B1 and B2 in masa flour, an alkaline cooked corn product

A comparison study of different extraction and clean up procedures for the liquid chromatographic analysis of fumonisins B1 (FB1) and B2 (FB2) in corn masa flour has been performed. They included extraction (heat or room temperature) with acidic conditions or EDTA containing solvents, and clean up by immunoaffinity or C18 solid phase extraction columns. Thereafter an analytical method has been optimized using extraction with an acidic mixture of methanol-acetonitrile-citrate/phosphate buffer, clean-up through immunoaffinity column and fumonisins determination by high performance liquid chromatography with automated pre-column derivatization with o-phthaldialdehyde reagent. Recovery experiments performed on yellow, white and blue masa flours at spiking levels of 400, 800 and 1200 μg kg-1 FB1, and 100, 200 and 300 μg kg-1 FB2 gave overall mean recoveries of 99% (± 6%) for FB1 and 88% (± 6%) for FB2. Good recoveries (higher than 90% for both FB1 and FB2) were also obtained with corn tortilla chips. The limit of quantification of the method (signal to noise ratio of 10) was 25 μg kg-1 for FB1 and 17 μg kg-1 for FB2. The method was tested on different commercial corn masa flours as well as on white and yellow corn tortilla chips, showing fumonisins contamination levels (FB1 + FB2) up to 1800 μg kg-1 (FB1 + FB2) in masa flour and 960 μg kg-1 in tortilla chips. Over 30% of masa flours originated from Mexico exceeded the EU maximum permitted level.


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