Xb c18 analytical column

Quantitation of ZEN and ZELs was performed by the method of Visconti and Pascale [31 (link)] with minor modifications. The analyses applied an HPLC pump (model 510, Waters, Milford, MA, USA) to drive eluent through an injector (Rheodyne 7125) supplied with a 20-μL sample loop onto a SecurityGuard Catridge (C18 4.0 × 3.0 mm, Phenomenex, Torrance, CA, USA) coupled to a Kinetex XB-C18 analytical column (250 × 4.6 mm, 5 μm particle size, Phenomenex, Torrance, CA, USA). The mobile phase contained acetonitrile (VWR), distilled water, and methanol (VWR): 46:46:8 v/v%. After injecting 20 μL samples, the isocratic elution was performed at room temperature with a 1 mL/min flow rate. Since the examined mycotoxins are fluorescent molecules [27 (link),32 (link)], ZEN and ZELs were quantified by a fluorescent detector (Jasco FP-920; λ_ex = 274 nm, λ_em = 440 nm). Data were recorded and evaluated using the Millennium Chromatography Manager (Waters, Milford, MA, USA), which also controlled the HPLC pump.

The analysis and purification of the crude compounds were carried out on reversed-phase high performance liquid chromatography (RP-HPLC) using Phenomenex Jupiter columns (4.6 × 250 mm, C18, 5 μm) and (21.2 × 150 mm, C18, 5 μm) with solvent A consist of 0.1% TFA in water, while solvent B consist of 0.1% TFA in 100% acetonitrile. All compounds were analyzed and purified by on a 5 μm XB-C18 analytical column (250 Å–4.6 mm, Phenomenex) on Waters system composed of 600-pump controller and 996-diode array detector. Final compounds such as 1, 1a, and 2 were purified on a 5 μm XB-C18 preparative column (150 Å–21.2 mm, Phenomenex) before to be tested. Fractions collected from the RP-HPLC were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) on a Bruker Daltonics Autoflex II TOF/TOF. α-cyano-4-hydroxy-cinnamic acid and sinapinic acid were the matrices utilized for characterization.