Ht 2 toxin

ZEN (100 µg/mL, 2 mL), T-2 toxin (100 µg/mL, 1 mL), and HT-2 toxin (100 µg/mL, 1 mL) were obtained from Sigma (St Louis, MO, USA). DON (B-trichothecenes mix 2, 100 µg/mL, 5 mL), AFs (AFs mix 1, 2 µg/mL for AFB₁ and AFG₁, and 0.5 µg/mL for AFB₂ and AFG₂, 5 mL), OTA (10 µg/mL, 5 mL), and FB₁ and FB₂ (FUMs mix 3, 50 µg/mL, 5 mL) were purchased from Biopure (Getzersdorf, Austria). Phosphate-buffered saline (PBS, pH 7.4) was purchased from Sigma. HPLC-grade acetonitrile and methanol were supplied by Merck (Darmstadt, Germany). Ammonium acetate (HPLC grade, 99.0%, Netherlands) and formic acid (mass spectrometry grade, 98%, St. Louis, USA) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). For purification, a Myco6in1+ IAC column obtained from Vicam was used (Boston, MA, USA). A Myco6in1+ column is intended to simultaneous detect the multiple mycotoxins including AFs, ochratoxin, FUMs, ZEN, DON, NIV, T-2, and HT-2 toxin.

Methanol (MeOH), acetonitrile water, and formic acid (FA) for LC mobile phase (HPLC grade) were purchased from Merck (Darmstadt, Germany). Sodium chloride (NaCl), ammonium formate (NH₄HCO₂), magnesium sulfate (MgSO₄), and octadecyl carbon chain-bonded silica (C18) were acquired from Sigma Aldrich (Milan, Italy). Eighteen mycotoxin standards (purity >98%) including aflatoxins (AFB1, AFB2, AFG1, and AFG2), zearalenone (ZEN), α-zearalanol (α-ZAL), α-zearalenol (α-ZEL), β-zearalanol (β-ZAL), neosolaniol (NEO), T-2 toxin, HT-2 toxin, enniatins (A, A1, B, and B1), alternariol monomethyl ether (AME), and alternariol (AOH) were obtained from Sigma-Aldrich (Milan, Italy).
Stock solutions of each mycotoxin were built by dissolving 1 mg of solid reference standard in 1 mL of methanol. An intermediate mixed solution containing all the mycotoxins at a concentration of 30 μg/mL was obtained after mixing individual stock solutions and diluting in MeOH:H₂O (70:30 v/v) 0.1% formic acid. Working standard solutions at 1.6, 0.4, 0.08 μg/mL were used for spiking experiments (fortification levels at 20, 5, and 1 µg/kg). All solutions were stored in safe conditions at −20 °C in screw-capped glass vials.

Solid crystals of 4,15-DAS, T-2 toxin, HT-2 toxin and NES were purchased from Sigma-Aldrich (St. Louis, MO, USA). Each compound was dissolved in acetonitrile (100 mg/L each) and the stock solution was stored at −30 °C. LC-MS-grade acetonitrile and water, and reagent-grade acetonitrile and ammonium acetate were purchased from Wako Pure Chemicals (Osaka, Japan). The retail food was purchased randomly from local supermarkets and small retail shops in Japan from the spring of 2016 to the winter of 2017. Fusarium strains were isolated in 2013 from soybean or azuki bean purchased from retail shops in Japan. These strains were morphologically identified as Fusarium avenaceum, Fusarium oxysporum, Fusarium proliferatum, Fusarium incarnatum-equiseti species complex (FIESC) or Fusarium sp.