Aflatoxins

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).

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.

Methanol and acetonitrile were HPLC grade (>98%), purchased from J.T. Baker (Xastoloc, Mexico). Aflatoxins and resveratrol standards were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

The stock solution of aflatoxins was purchased from Sigma-Aldrich (St Louis, MO, USA) and consisted of a mix with 1 µg AFB1, 0.3 µg AFB2, 1 µg AFG1, and 0.3 µg AFG2 in methanol. Calibration curves for aflatoxins B1, B2, G1, and G2 were prepared at concentrations of 0.5, 1, 2.5, 5, and 10 ng/mL for AFB1 and AFG1 and 0.25, 0.5, 0.7, 1.5, and 3 ng/mL for AFB2 and AFG2. An intermediate solution for aflatoxins was made by 100-fold dilution of the original mix; then, working calibration solutions were prepared with mobile phase, stored at 4 °C, and renewed every week. Stock and intermediate standard solutions were stored at –20 °C. The immunoaffinity column (IAC) AflaTest WB SR was acquired from VICAM (Watertown, MA, USA). Deionized water was obtained from a Millipore milli-Q water purification system (Mildford, MA, USA). HPLC-grade acetonitrile and methanol were supplied from Scharlau (Scharlab, Barcelona, Spain), and sodium chloride was purchased from Panreac (Barcelona, Spain).
As a safety note, all used laboratory glassware were treated with an aqueous solution of sodium hypochlorite (5%) before discarding to minimize health risks due to mycotoxin contamination [18 (link)].