Zearalanone

The individual mycotoxin solid calibration standards (1 mg) of DON, 3-ADON, 15-ADON, DOM-1, NIV and zearalanone (ZAN, internal standard) were purchased from Sigma-Aldrich (Bornem, Belgium). DON-3G (50.2 ng/µL, in acetonitrile) was purchased from Biopure Referenzsubstanzen (Tulln, Austria).
All mycotoxin solid standards for LC-MS/MS analysis were dissolved in methanol (1 mg/mL), and were storable for a minimum of 1 year at −18 °C [60 (link)]. DON-3G was kept at 4 °C. The working solutions of DON, 3-ADON, 15-ADON, DOM-1 and NIV (10 ng/µL) were prepared in methanol, and stored at −18 °C, while DON-3G (10 ng/µL) was dissolved in acetonitrile (4 °C, monthly renewed).
DON standard for the bioassay and the enrichment protocol was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL and stored at −18 °C. Working solutions of 100 and 10 mg/L DON were prepared in “Lemna medium” or “minimal incubation medium” (MMO) and stored at −18 °C.

Reference standards of Zen (≥ 99%), Zer (≥ 98%, HPLC grade), zearalanone (Zan; ≥ 98%), β-zearalanol (bZal; ≥ 96%, HPLC grade), α-zearalenol (aZol; ≥ 98%), and β-zearalenol (bZol; ≥ 97%) were obtained from Sigma-Aldrich. The internal standards Rac-Zearalenone-d₆ (Zen-d₆; ≥ 96.9%) and α-zearalenol-d₇ (aZol-d₇; ≥ 98%) were obtained from Toronto Research Chemicals. Sodium acetate buffer solution (pH = 4.65, 0.2 M) was purchased from Honeywell Fluka. The enzyme β-glucuronidase from Helix pomatia (type HP-2, > 100,000 units/mL, sulfatase activity < 7500 units/mL) was obtained from Sigma-Aldrich. HPLC-grade methyl tert-butyl ether (MTBE) and methanol (MeOH) were purchased from VWR. HPLC-grade acetonitrile (ACN) was purchased from Honeywell. Water was obtained from a Milli-Q water purification system (Millipore).

Mycotoxin standards comprising of aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), aflatoxin G₁ (AFG₁), aflatoxin G₂ (AFG₂), fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), deepoxy-deoxynivalenol (DOM), 15-acetyl-deoxynivanelol (15-ADON), neosolaniol (NEO), OTA, alternariol (AOH), alternariol monomethyl ether (AME), zearalenone (ZEN), nivalenol (NIV), deoxynivanelol (DON), 3-acetyl-deoxynivanelol (3-ADON), sterigmatocystin (STE), roquefortine C (ROQ C), enniatin B (ENN B), fusarenon-X (FUS-X), HT-2 toxin (HT-2) and zearalanone (ZAN) were obtained from Sigma-Aldrich (Bornem, Belgium). Fumonisin B₃ (FB₃) was procured at Promec Unit (Tynberg, South Africa), while T-2 toxin (T-2) and diacetoxyscirpenol (DAS) were purchased from Biopure Referenzsubstanze (Tulln, Austria).

The (4-acrylamidophenyl)(amino)methaniminium acetate (AAB) was synthesized as previously described [34 (link)]. The 4-vinylpyridine (4-VPY), methacrylamide (MAM), ethylene glycol dimethacrylate (EGDMA), azo-bis-dimethylvaleronitrile (ABDV), azobisisobutyronitrile (AIBN), zearalanone (ZAN), zearalenone (ZON), hyaluronidase, N-acetylglucosaminidase, hyaluronic acid-binding protein (HABP), biotin, streptavidin, and fluorescein-5-isothiocyanate (FITC) was purchased from Sigma-Aldrich (Shanghai, China). Fluorescein dimethacrylate (PolyFluor^® 511) was obtained from Polysciences (Hirschberg an der Bergstrasse, Germany). Glucuronic acid (GA), tetracycline (TC), amoxicillin (AMO), and levofloxacin (LEV) came from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the solvents used in the experiments as well as acids and bases are of analytical grade and anhydrous for acetonitrile (ACN) and dimethylsulfoxide (DMSO). The materials for cells culturing are: Phosphate buffer saline (PBS), penicillin/streptomycin, high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplied with L-glutamine, fetal bovine serum (FBS), trypsin, ethylenediaminetetraacetic acid, and were obtained from Thermo Fisher Scientific (Shanghai, China).

FB₁, FB₂, DON, deepoxy-deoxynivalenol (DOM), NIV, ZEN, and zearalanone (ZAN) standards were purchased from Sigma-Aldrich (Bornem, Belgium), while FB₃ was from Promec Unit (Tynberg, South Africa). LC-MS grade methanol, glacial acetic acid, analytical-grade acetonitrile (Biosolve B. V., Valkenswaard, The Netherlands), analytical-grade methanol, ammonium acetate, formic acid, calcium hydroxide, calcium chloride, sodium hydroxide, potassium hydroxide (Merck, Darmstardt, Germany), dichloromethane, ethyl acetate (Acros Organics, Geel, Belgium), and n-hexane (VWR International, Zaventem, Belgium) were used including ultra-pure water from Arium^® pro Ultrapure Water System (Sartorius, Goettingen, Germany). Wood ashes were collected within Ghent environment, Belgium, while GracePure aminopropyl (NH₂) solid-phase extraction (SPE) 1000 mg/6 mL cartridges were obtained from Grace Discovery Sciences (Lokeren, Belgium).

The individual mycotoxin solid calibration standards (1 mg) of aflatoxin B1(AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), and Zearalanone (ZAN) (internal standard) were obtained from Sigma Aldrich (Bornem, Belgium) Zearalanone was used as internal standard to quantify the AFs, but also to feature as identification criterium for the other metabolites. One of the identification criterium is the relative retention time. The retention time of the internal standard is compared to the retention time of the metabolites, and should remain constant.
All mycotoxin solid standards were dissolved in methanol (1 mg/mL) and were storable for a minimum of 1 year at −18°C (Spanjer et al., 2008 (link)). The working solutions of AFB1, AFB2, AFG1, AFG2, and ZAN (10 ng/μl) were prepared in methanol, and stored at −18°C and renewed monthly. Water was obtained from a Milli-Q® SP Reagent water system from Millipore Corp. (Brussels, Belgium). Disinfectol® (denaturated ethanol with 5% ether) was supplied by Chem-Lab (Zedelgem, Belgium). Methanol (LC-MS grade) was purchased from BioSolve (Valkenswaard, the Netherlands), while acetonitrile (Analar Normapur) and ammonium acetate were obtained from VWR International (Zaventem, Belgium). Acetic acid (glacial, 100%) was supplied by Merck (Darmstadt, Germany).

Zearalenone (ZEN; MW = 318.36 g/mol; purity: 99.7%, HPLC), zearalanone (ZAN; MW = 320.38 g/mol; purity: 100%, TLC ), α-zearalanol (α-ZAL; MW = 322.40 g/mol; purity: 98%, HPLC), β-zearalanol (β-ZAL; MW = 322.40 g/mol; purity: 98%, HPLC), HSA (MW = 66.4 kDa), BSA (MW = 66.4 kDa), PSA (MW = 67.5 kDa), RSA (MW = 64.6 kDa), and warfarin (MW = 308.3 g/mol) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Zearalenone-14-sulfate ammonium salt (Z14S; MW = 415.46 g/mol; purity: 98.5%, HPLC, NMR, LC-MS) was purchased from ASCA GmbH (Berlin, Germany). Zearalenone-14-O-β-d-glucoside (Z14G; MW = 480.50 g/mol; purity: 99.4%, HPLC, NMR) was obtained from Honeywell (Charlotte, NC, USA). Stock solutions of mycotoxins (5000 μmol/L; ZEN: 1.592 g/L; ZAN: 1.602 g/L; ZALs: 1.612 g/L; Z14S ammonium salt: 2.078 g/L; and Z14G: 2.403 g/L) were prepared in ethanol (96 v/v%, spectroscopic grade; VWR, Debrecen, Hungary) and stored at – 20 °C.