Mycotoxin Extraction and Purification

Extractions of mycotoxins from samples were prepared as described in previous reports [21 (link),22 (link)]. Briefly, 25 g of the ground samples were mixed with 100 mL of methanol:water (80:20, v/v) for AFB₁ measurement, acetonitrile:water solution (84:16, v/v) for ZEN measurement or methanol:water (60:40, v/v) for DON measurement. The samples were blended at high speed for 3 min, and then filtered through Mycosep^® #226 (Romer Labs. Inc., Singapore). The solvent extracts were diluted with phosphate-buffered saline solution (PBS, pH 7.4), then filtered through immunoaffinity columns; ZearaStar (Romer Labs, Tulln, Austria) for ZEN, AokinImmunoClean CF AFLA and CF DON (Aokin AG, Berlin, Germany) for AFB₁ and DON, respectively. After column washing with PBS and a methanol-water solution, the mycotoxins were eluted from the columns with methanol, and mycotoxins concentrated to dryness under anitrogenair steam. The mycotoxin residues were then immediately re-dissolved in a mobile phase described below, filtered through a Millex PTFE 0.22 μm filter (Merck, Tianjin, China), and analyzed by high-performance liquid chromatography (HPLC).