The mixture sample solution was made
with 5 mL of sample filtrate diluted with 4 mL of 4.5Msodium
acetate buffer at pH 6.2, and 2 mL of 1,2-phenylenediamine
solution (2.5 mg dissolved in 25 mL of deionized water) and
allowed to stand at 37 °C during 30 min in the dark. After
that time, the solution was cooled and injected onto the HPLC