Ochratoxin a

Human serum albumin (HSA), bovine serum albumin (BSA) and ovalbumin (OVA), Sodium azide, glycine, Tween-20, Triton X-100, and morpholinoethanesulfonic acid (MES) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carboxyl magnetic beads (10 mg/mL) were purchased from Kangyuan Techbio Biologicals (Beijing, China). Ultra-pure water used for the preparation of all the reagents was obtained from a Milli-Q purification system (Bedford, MA, USA). A standard solution of ochratoxin A (OTA), aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), aflatoxin G₁ (AFG₁), aflatoxin G₂ (AFG₂), HT-2 toxin (HT-2), nivalenol (NIV), sterigmatocystin (ST), zearalenone (ZEN), T-2 toxin (T-2), Deoxynivalenol (DON), deoxynivalenol-3-glucoside (3G-DON), 3-acetyl-deoxynivalenol (3-AcDON), and 15-acetyl-deoxynivalenol (15-AcDON) were purchased from Biopure (Tulln, Austria) and were stored at −18 °C. Working solutions were prepared by dilution with HPLC-grade methanol and then stored in vials at 4 °C and renewed weekly. HPLC-grade methanol (MeOH) and Acetic acid (HAC) were purchased from Fisher Scientific (Atlanta, GA, USA).

Citrinin (solution in acetonitrile 100 μg mL⁻¹), ochratoxin A (solution in acetonitrile 100 μg mL⁻¹) and aflatoxin mix containing AFB1 and AFB2 (2 μg mL⁻¹ in acetonitrile), AFG1 and AFG2 (0.5 μg mL⁻¹ in acetonitrile) were obtained from Romer Labs (Tulln, Austria). Monacolin K (25 mg), monacolin K acid (5 mg) and simvastatin (10 mg) were purchased from Vinci Biochem S.R.L. (Florence, Italy).
HPLC-grade ethanol, and acetonitrile were purchased from Sigma-Aldrich (Taufkirchen, Germany); bidistilled water was obtained using Milli-Q System (Millipore, Bedford, MA, USA). MS-grade formic acid from Fisher Chemical (Thermo Fisher Scientific Inc., San Jose, CA, USA) was also used.

Mycotoxin standard solutions of aflatoxins B1, B2, G1 and G2, fumonisin B1 and B2, A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyl-deoxynivalenol, fusarenone X, diacetoxyscirpenol, T-2 toxin, HT-2 toxin), zearalenone, ochratoxin A, and patulin were obtained from Romer Labs (Tulln, Austria). All the solvents applied for both the extraction and analysis steps, methanol, acetonitrile formic acid and acetic acid, were HPLC-grade and were purchased from Sigma-Aldrich (Milan, Italy), while Bi-distilled water was produced in-house by using a Milli-Q System (Millipore, Bedford, MA, USA). Salts used for extraction as for the preparation of the eluents as sodium chloride and ammonium acetate were obtained from Sigma-Aldrich (Milan, Italy).

Piglets received a grower diet consisting of a mash feed based on wheat, soy, and barley as specified in Table 6.
The diet was analyzed for the most relevant mycotoxins, i.e., ZEN, deoxynivalenol, ochratoxin A, fumonisins B1 and B2, as well as aflatoxins B1, B2, G1, and G2 by Romer Labs GmbH (Tulln, Austria) using HPLC-MS/MS analysis. Concentrations of all mycotoxins were below the limit of detection.

Standards of aflatoxin B₁ (AFB₁), aflatoxin G₁ (AFG₁), sterigmatocystin (STER), zearalenone (ZEN), and ochratoxin A (OTA) were supplied by Romer Labs (Beijing) Co., Ltd. The purity of standards was declared in the range 95%–98.9%. The standards dissolved in acetonitrile were stored at −20℃ in amber glass vials.
Acetonitrile (ACN, HPLC grade), methanol (MeOH, HPLC grade), formic acid (MS grade), and ammonium acetate (MS grade) were purchased from Merk Corp. Anhydrous magnesium sulfate, trisodium citrate dihydrate, disodium hydrogen citrate sesquihydrate, sodium chloride, sodium acetate, chromium nitrate nonahydrate, and terephthalic acid were from Aladdin Co. Absolute ethyl alcohol, N, N‐dimethylformamide, hydrochloric acid, and ammonium hydroxide were from Chengdu Kelin Chemical Industry Co. Deionized water was prepared by a Milli‐Q system (Millipore Co.).

Analytical standards of mycotoxins were purchased from Romer Labs (Tulln, Austria) including: aflatoxin B₁ (AFB₁), zearalenone (ZEN), ochratoxin A (OTA), deoxynivalenol (DON), nivalenol (NIV), fumonisin B₁ (FB₁) and T-2 toxin (T-2). All standards were dissolved in LC-MS grade acetonitrile to the initial concentration of 100 µg/mL. All mycotoxin solutions were prepared immediately prior to the binding tests.
Other reagents used in this study were: LC-MS-grade metanol (Rathburn Chemicals Ltd., Walkerburn, UK); LC-MS-grade water (Merck, Darmstadt, Germany); LC-MS-grade formic acid (FA) and ammonium formate (Sigma Aldrich, St. Louis, MO, USA); HPLC-grade isopropyl alcohol (Merck, Darmstadt, Germany), analytical grade: glycerol (99.5%), ethanol (96%), NaCl, NaOH, H₂SO₄ (95%), Tris-HCl, KCl, Na₂HPO_4, KH₂PO₄, glacial acetic acid, sodium lauryl sulfate (Avantor Performance Materials, Gliwice, Poland); 3,5-Dinitrosalicylic acid (Sigma-Aldrich, USA); Pronase E (Sigma-Aldrich, USA); Zymolyase 20T (MP Biomedicals LLC); Yeast Beta-Glucan Kit, K-EBHLG (Megazyme, Ireland); zirconium-glass beads of 1 mm (Biospec Products, USA); high retention cellulose tubing bags for dialysis (Sigma-Aldrich, St. Louis, USA); 0.2-µm mesh, 4-mm-diameter nylon syringe filters purchased from Phenomenex (Torrance, CA, USA).

HPLC grade acetonitrile, methanol, water and formic acid were purchased from Fisher Scientific. Japanese aflatoxin mixture (25 μg/mL) from Sigma-Aldrich; deoxynivalenol (25 µg/mL), nivalenol (25 µg/mL), zearalenone (10 µg/mL), ochratoxin A (10 µg/mL) from Romer Labs; diacetoxyscirpenol (100 µg/mL), T-2 toxin (100 µg/mL), HT-2 toxin (100 µg/mL), fumonisin B1 (100 µg/mL), fumonisin B2 (100 µg/mL), and fumonisin B3 (100 µg/mL) from Trilogy. QuEChERS EN extraction salts and Captiva EMR-Lipid cartridge were acquired from Agilent (CA, USA).