Cholesterol content was determined according to the method of
Piironen et al [19], with some modification. Extracted fat from
sample was followed with the mixture (60% KOH 8 mL and alcohol
40 mL) by 1 hour of at 100C, followed by 60 mL of mixed solution
for cooling. The application of 50 mL of benzene and 1N KOH 100
mL separates the supernatant, which was added by 0.5N KOH 20
mL and anhydride sodium sulfate. Sample solution was then
analyzed by GC (Hewlett Packard 6890 series) after decompressed
enrichment and application of 5a cholestane solution as an internal
standard solution. The column was HP-1 capillary column (30 m
0.32 mm internal diameter, 0.25 mm film thickness), detector
(initial temp. 200C (1 min)), oven temp. (increase rate 20C/min,
final temp. 300C (6 min)), injector temp. 250C, detector temp.
300C, helium carrier gas, and flow rate of 1 mL/min