4.3.4. Modified QuEChERS Method (method D)

A corn sample (5 g) was weighed into a 50 mL centrifuge tube with 10 mL of water containing 0.3% formic acid and 10 mL of acetonitrile. After a brief vortex, the mix was placed in Geno/Grinder for 20 min to homogenize well. QuEChERS salts were added and shaken for 2 more minutes on a Geno/Grinder homogenizer and then centrifuged for 7 min. Subsequently, 1.6 mL of the extract was diluted with 0.4 mL of water. The diluted sample was passed through a lipid removal under constant vacuum. The extract was evaporated to dryness at 40–50 ◦C under a gentle stream of nitrogen and reconstituted in mobile phase A/mobile phase B (50/50, v/v).

This modified QuEChERS method uses an acidic condition to increase the extraction efficiency by using water with 0.3% formic acid. The polarity of compounds can be changed by using different pH levels [28]. It also offers evaporation/reconstitution for improving the MS responses and peak broadening. However, nivalenol cannot be completely extracted in acetonitrile; acidic condition and compatibility with mobile phases are essential in this procedure. The presence of the 0.3% formic acid improves the analyte partitioning into the organic phase and its elution as the first compound in the chromatographic run. Moreover, it prevents the retention of Ochratoxin A on the lipid removal cartridge and provides better results. Finally, the salting-out step with anhydrous MgSO<sup>4</sup> and NaCl followed by lipid removal and reconstitution steps achieved highest recoveries.
