The ethyl acetate phase was washed with distilled water
twice. The ethyl acetate extracts were diluted in 100-mL each
of methanol and n-hexane to remove fatty acids (polar elements)
and other nonpolar elements. The solvents were then
separated using a Buchner vacuum separation funnel. The solvents
were evaporated at 40 C on a rotary evaporator–shaker
until a 5-mL solvent volume was reached; then this sample was
immediately used for GC–MS analysis or stored in the deep
freezer at 20 C. Each solvent was subjected to three replications
in the experiments.