The
hydrolyzed sample was cooled on ice, concentrated using the
Vivaspin2 (GE Healthcare – 30 kDa) and centrifuged for
10 min at 5000 g. From the hydrolyzed filtrate, 100 mL was
transferred to test tubes containing the same volume of the
DMB reagent, and the solution was heated at 80 1C for
50 min. The derivatized samples were cooled on ice and
diluted with the addition of 800 mL of ultrapure water.