The extraction and quantification of cholesterol were
carried out by the method published by Al-Hasani,
Hlavac, and Carpenter (1993). Sardine samples (10:000
0:001 g) were transferred to a 250 ml flat-bottom flask.
The sample was stirred in an ethanol–methanol–isopropanol
(90:5:5, v/v/v) solution, in an amount equivalent to
4 ml/g sample, and 1 ml 60% KOH/g sample. The flask
containing the mixture was connected to a water-cooled
condenser, and refluxed for 1 h. After cooling the digest
to room temperature, 100 ml of hexane was added, and
the mixture was stirred for 10 min. Next, 25 ml of deionized
water were added and the mixture was stirred for
further 15 min.