The optimization condition was performed
using an isocratic elution at the proportion of
60:40 (v/v) 5 mM ammonium acetate aqueous
solution adjusted to pH 4.4: methanol at a
flow rate of 1 mL/min. The C18 column
with 5 cm length was preferred as the
complete separation was occurred within 3
min and the mobile phase operated was also
3 times decreased.