Nivalenol

Ergosterol (ERG), fumonisin B₁ (FB₁), zearalenone (ZON), deoxynivalenol (DON), and nivalenol (NIV) standards were purchased with a standard grade certificate (purity above 98%) from Sigma-Aldrich (Steinheim, Germany). Stock solutions of standards were prepared in acetonitrile except ERG in methanol at 1.0 mg/mL concentrations and stored at −20 °C. Sodium dihydrophosphate, potassium hydroxide, sodium hydroxide, potassium chloride, acetic acid, hydrochloric acid, and o-phosphoric acid were purchased from POCh (Gliwice, Poland). Organic solvents (HPLC grade), disodium tetraborate, n-pentane, 2-mercaptoethanol, sodium acetate, and all the other chemicals were also purchased from Sigma-Aldrich (Steinheim, Germany). Water for the HPLC mobile phase was purified using a Milli-Q system (Millipore, Bedford, MA, USA).

HPLC grade solvents (hexane, methanol (MeOH) and acetonitrile (AcN)) were purchased from Merck (Darmstadt, Germany). Deionized water (<18 MΩ cm resistivity) was obtained in the laboratory using a Milli-Q SP Reagent Water System (Millipore, Bedford, MA, USA).
Standards of mycotoxins including (aflatoxins (B1, B2, G1 and G2); enniatins (A, A1, B and B1); fumonisins (B1 and B2); nivalenol (NIV); deoxynivalenol (DON); 15-acetyldeoxynivalenol (15-ADON); 3-acetyldeoxynivalenol (3-ADON); zearalenone (ZEA); beauvericin (BEA); fusarenon X (FUS-X)) were purchased from Sigma Aldrich (Madrid, Spain). The T-2 and HT-2 toxins were provided from BiopureReferenzsubstanzenGmBH (Tulln, Austria). Finally, fumonisin B3 was provided by the Research Program “PROMEC” (Tygerberg) in South Africa. All stock solutions of mycotoxins standards were stored in glass-stoppered bottles in darkness at −20 °C until the final analysis. For the derivatization, the reagent composed of BSA (N,O-bis (trimethylsilyl) acetamide)/TMCS (trimethylchlorosilane)/TMSI (N-trimethylsilyimidazole) (3:2:3) was obtained from Supelco (Bellefonte, PA, USA).

Deoxynivalenol (DON), nivalenol (NIV) and fusarenon X (FX) were purchased from Sigma (Saint-Louis, MO, USA). Stock solutions of mycotoxins were performed in methanol to reach 100 mM DON, 10 mM NIV and 100 mM FX. Working dilutions were prepared in cell culture media without FCS and antibiotics. Mycotoxin stock solutions as well as working dilutions were stored at −20 °C. Methanol at 0.2%, corresponding to the highest concentration reached upon cell exposure to mycotoxins, had no impact on cell viability and cytokine production. Methanol (LC-MS grade) was obtained from Carlo Erba Reagents (Val De Reuil, France).