Lichrocart precolumn

Deacetylation was monitoring both in term of disappearance of 3- or 15ADON and formation of DON. Measurement of DON, 3ADON and 15ADON concentrations was performed by reversed-phase HPLC using Waters Alliance System equipped with a Waters 2690 XE separation module and a Waters 996 photodiode array detector (Waters, Saint Quentin Falavier, France). The separation was achieved by mean of a LiChroCart column (125 mm × 4 mm) packed with 5 μm RP-18 Purospher gel (Merck, Darmstadt, Germany) and equipped with a LiChroCart precolumn (4 mm × 4 mm) packed with the same material (Merck). The precolumn and column were placed in room temperature. The elution procedure was carried out at 0.7 mL·min⁻¹ by a methanol–water solvent, using the following conditions: 40% methanol during 2 min followed by an increase in methanol to 50% at 2.1 min. The detection wavelength was set at 237 nm allowing the best signal/baseline ratio for simultaneous detection of the three DON forms in the incubation mediums. Under these conditions, DON and 3/15ADON were clearly separated, the retention time being around 2.7 min and 5.7 min, for DON and 3/15ADON, respectively (Figure 9). Quantification was based on calibration with the corresponding standard using Millinium Software (Waters, Saint Quentin Falavier, France).