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4 protocols using fumonisin b2

1

Fumonisin Quantification in Food Samples

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Solvents used for the sample preparation and HPLC-FLD analysis were all HPLC-grade, while solvents used for the HPLC-HRMS analysis were MS-grade. Acetonitrile (ACN), ammonium formate, formic acid, ethyl acetate, MeOH, and trifluoroacetic acid were all purchased from Sigma-Aldrich (Schnelldorf, Germany), while 2-BuOH was purchased from Fisher Scientific (Waltham, MA, USA). Water was purified by reverse osmosis and a subsequent MilliQ-system (Millipore, Bedford, MA, USA). Fluorescence derivatization was achieved using AccQ-Fluor reagent WAT052880 (Waters, Milford, MA, USA). External calibration was carried out using a fumonisin B1 (49.9 µg/mL) and fumonisin B2 (50.6 µg/mL) mixture in H2O:ACN (1:1, v/v), purchased from Romer Labs (Tulln, Austria). Four different Isolute SPE cartridges (C18 (MFC) (3 mL, 500 mg), C8 (3 mL, 500 mg), C2 (3 mL, 500 mg) and Isolute Myco (3 mL, 60 mg)) were purchased from Biotage (Uppsala, Sweden), while Strata-X (3 mL, 30 mg) SPE cartridges were purchased from Phenomenex (Torrance, CA, USA).
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2

Stable Isotope-Labeled Internal Standards for Mycotoxin Analysis

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There were 15 stable isotope labeled internal standards: [13C17]-aflatoxin B1 (500 ng/mL), [13C17]-aflatoxin B2 (500 ng/mL), [13C17]-aflatoxin G1 (500 ng/mL), [13C17]-aflatoxin G2 (500 ng/mL), [13C15]-deoxynivalenol (25,000 ng/mL), [13C34]-fumonisin B1 (25,000 ng/mL), [13C34]-fumonisin B2 (10,000 ng/mL), [13C20]-ochratoxin A (10,000 ng/mL), [13C24]-T-2 toxin (25,000 ng/mL), [13C22]-HT-2 toxin (25,000 ng/mL), [13C18]-zearalenone (25,000 ng/mL), [13C17]-3-acetyl-deoxynivalenol (25,000 ng/mL), [13C17]-15-acetyl-deoxynivalenol (10,000 ng/mL), [13C19]-diacetoxyscirpenol (25,000 ng/mL) and [13C15]-nivalenol (25,000 ng/mL) were purchased from Biopure (Romer Labs, Tulln, Austria).
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3

Sample Preparation and Analysis Protocol

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All solvents used for sample preparation were of HPLC grade and purchased from Sigma-Aldrich (Schnelldorf, Germany). All solvents used for sample analysis, water (H2O), methanol (MeOH), and acetonitrile (ACN), were of LCMS grade and purchased from Sigma-Aldrich. Formic acid (≥96%) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). H2O for SPE was purified on a Milli-Q system (Merck Millipore, Billerica, MA, USA). Iturin A2 (>95%) was purchased from Sigma-Aldrich and the stock solution (20 μg·mL−1) was prepared in HPLC grade MeOH. Sodium bicarbonate (NaHCO3), 98% 13C-labelled, was purchased from Sigma-Aldrich. Fluorescence derivatisation was achieved with AccQ-Fluor reagent WAT052880 (Waters; Milford, MA, USA). The mixture of fumonisin B1 (49.9 μg·mL−1) and fumonisin B2 (50.6 μg·mL−1) in H2O/ACN (1:1, v/v) was purchased from Romer Labs (Tulln, Austria). External standards valinomycin and chloramphenicol were purchased from Sigma-Aldrich. Five different, 3 mL, 30 mg, SPE cartridges, Strata-X, Strata-SCX, Strata-WCX, Strata-SAX, Strata-MAX, were purchased from Phenomenex (Torrance, CA, USA). Three millilitre, 30 mg Oasis-HLB SPE cartridges were purchased from Waters.
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4

Mycotoxin Immunoassay Reagent Development

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Antibodies for ZEA, T2 and FUM and mycotoxin-BSA conjugates for ZEA (3.39 mg/ml), T2
(1.5 mg/ml) and FUM (1.32 mg/ml) were provided by Tecna (Tecna s.r.l, Trieste, Italy). Nunc 96 well microtitre plates were purchased from VWR (Leicestershire, UK). Alkaline phosphatase substrate was purchased from Millipore (Hertfordshire, UK). Bovine serum albumin (BSA), anti-rabbit IgG-alkaline phosphatase antibody produced in goat, 5-bromo-4chloro-3'-indolyphosphate (BCIP), nitro-blue tetrazolium (NBT), methanol (HPLC grade), zearalenone, deoxynivalenol and HT2-toxin were all purchased from Sigma-Aldrich (Dorset, UK). Fumonisin B1 was purchased from Trilogy (Darmstadt, Germany). T2-toxin, fumonisin B2 and fumonisin B3 were purchased from Romer Labs (Cheshire, UK).
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