The residue from the extraction was saponified for 1 h
with 10 ml 10% methanolic potassium hydroxide, containing
0.1% BHT in the dark at room temperature, according
to Hart and Scott (1995). The carotenoids were extracted
from the methanolic potassium hydroxide phase with 20 ml
diethyl ether, containing 0.1% BHT, and 20 ml 10%
sodium chloride solution. The combined diethyl ether
phase was washed with water until neutral pH, dried over
anhydrous sodium sulfate and concentrated to dryness in a
rotary evaporator at 35 1C. The residue was dissolved in
acetonitrile:methanol:ethyl acetate (60:20:20, v/v/v). The
sample solution was filtered through a 0.45 mm Nylon
membrane filter (Millipore) and 20 ml were injected for
HPLC analysis.