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Certified standards of 4 aflatoxins (AFs: AFB₁, AFB₂, AFG₁, AFG₂), OTA, OTB, DAS, NIV, NEO, T-2, HT-2, DON, 3-AcDON, 15-AcDON, ZEN, α-ZOL, β-ZOL and fusarenon X (FUS-X) were obtained from Romer Labs Inc. (Union, MO, USA). ZAN and α-ZAL were purchased from Sigma-Aldrich (Buchs, Switzerland), and β-ZAL was purchased from Pribolab Private Limited (Immunos, Singapore).
The individual stock solutions of AFs, ZEN, T-2, OTA and OTB were dissolved in acetonitrile at 10 mg L⁻¹ and the other 13 standards were prepared in acetonitrile at 100 mg L⁻¹. AFs, ZEN, T-2, OTA and OTB were prepared in enough acetonitrile at 0.5 mg L⁻¹ as an intermediate mixed solution (Mix A). A similar procedure was carried out for Mix B including DAS, DON, FUS-X, HT-2, 3-AcDON, 15-AcDON, ZAN, α-ZAL, β-ZAL, α-ZOL and β-ZOL toxins, at a stock concentration of 10 mg L⁻¹. Mix C, of NEO and NIV at a concentration of 25 mg L⁻¹, was obtained in the same way. Then, 1 mL of each mixture, Mix A, Mix B and Mix C, and 7 mL acetonitrile, were mixed to obtain 10 mL of total mixed stock of the standard solution, which was stored at −20 °C in the dark. Working solutions were freshly diluted with the blank matrix or acetonitrile before UPLC–MS/MS analysis.

The AFB₁, AFB₂, AFG₁, DON, 3-AcDON, 15-AcDON, ZEN, β-ZEL, α-ZEL, β-ZAL, and α-ZAL standard solutions were purchased from Sigma-Aldrich (St. Louis, MO, USA) and stored at −20 °C before use. All organic solvents, salts, and acids were of analytical or HPLC grade. Acetonitrile, methanol, hexane, and isopropanol were purchased from Merck (Darmstadt, Germany). Ammonium acetate and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q quality water (Millipore, Billerica, MA, USA) was used throughout the experiments. An Agilent Extend-C18 column (100 mm × 4.6 mm, 3.5 μm) was obtained from Agela Technologies (Tianjin, China).

Water and methanol (MeOH) for LC mobile phase (LC-MS grade) and acetonitrile (ACN) were provided by Merck (Darmstadt, Germany). Ammonium formate (analytical grade) was supplied by Fluka (Milan, Italy). Formic acid (MS grade) was acquired from Carlo Erba reagents (Cornaredo, Italy). Sodium chloride (NaCl) and octadecyl carbon chain-bonded silica (C18) (analytical grade) and were provided by Sigma Aldrich (Milan, Italy). Conical centrifuge polypropylene tubes of 50 and 15 mL were provided by BD Falcon (Milan, Italy). Syringe filters with polytetrafluoroethylene membrane (PTFE, 15 mm, diameter 0.2 µm) were supplied by Phenomenex (Castel Maggiore, Italy).
Analytical standards of the following mycotoxins (HPLC purity > 98%): AFB1, AFB2, AFG1, AFG2, BEA, dDON, ENNA, ENNA1, ENNB, ENNB1, FB1, FB2, FUS-X, HT-2, NEO, OTA, T-2, α-ZEL, α-ZAL, β-ZEL, β-ZAL, ZAN and ZEN were purchased from Sigma-Aldrich (Milan, Italy).
For each analytical standard, a stock solution was prepared by dissolving 1 mg in 1 mL of MeOH. Afterwards, working solutions were built by properly diluting in MeOH/H₂O (70:30 v/v) 0.1% formic acid until reaching the desired concentrations for spiking experiments (50, 25 and 10 µg/kg). Working solutions were stored in securely closed vials at −20 °C.

The standards of AFB₁, AFB₂, AFG₁, AFG₂, OTA, FB₁, FB₂, ENA, ENA₁, ENB, ENB₁, BEA, STG, ZON, α-ZAL, β-ZAL, α-ZOL, β-ZOL, DON, 3-ADON, 15-ADON, DAS, NIV, FUS-X, NEO, T-2, and HT-2 toxins were purchased from Sigma Aldrich. Individual stocks of all analytes were prepared to obtain 20 mg/L in methanol and multianalyte working solutions of 2 mg/L were also used by diluting the individual stock solutions. The multianalyte working standard solution was used for standard calibration curves, matrix-matched calibration curves, and recovery assays. All standards were stored in darkness and kept at −20 °C.

The mycotoxin standards of ZEN, α-ZOL, β-ZOL, α-ZAL, β-ZAL, and ZAN were purchased from Sigma-Aldrich (St. Louis, MO, USA). The standards of Z14G, α-ZOL14G, and β-ZOL14G were kindly provided by the Laboratory of Food Analysis, Ghent University (Belgium). Methanol and acetonitrile (HPLC-grade) were purchased from Merck (Darmstadt, Germany). Ultrapure water (18.2 MΩ·cm) used in our experiments was obtained from a Milli-Q System (Bedford, MA, USA). Cleanert MC clean-up columns were purchased from Bonna-Agela Technologies (Tianjin, China). Other chemicals were obtained from Aladdin (Shanghai, China).

Standard mycotoxin powders, including ZEN, ZAN, α-ZAL, β-ZAL, α-ZEL, β-ZEL (1 mg dissolved in 1 mL acetonitrile for 1 mg/mL, stored at −20 °C for one year), ¹³C₁₈-ZEN ISTD (25.2 µg/mL in acetonitrile, stored at −20 °C for one year), and Sephrose 4B gel (CNBr-activated) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Toluene, HPLC-grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). Bis(trimethylsilyl) trifluoroacetamide with 1% Trimethylchlorosilane (BSTFA+1%TMCS) was purchased from Anpel (Shanghai, China) for derivatization. A zearalenone monoclonal antibody was obtained from Clover Technology Group Inc. (Beijing, China). Other analytical-grade chemicals and reagents were obtained from Beijing Chemical Reagent Co. (Beijing, China).

HPLC grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Phosphate buffered saline(PBS) was purchased from Sigma (St. Louis, Mo, USA). ZEN(α-ZAL, β-ZAL, α-ZOL, β-ZOL, ZAN, ZON) were purchased from Sigma (St. Louis, Mo, USA). As purification IAC, immunoaffinity columns for Zearalenones (IAC-ZER) was purchased from Clover (Irvine, USA). All other organic chemicals and organic solvents were reagent grade or higher.