Briefly, IS (9.82 μg of 19-hydroxycholesterol)was added to the extracted
lipids. Cold saponification was performed at room temperature with
10 mL of methanolic 1 N KOH, in darkness and under continuous agitation
in an orbital shaker (IKA KS26, Stauffen, Germany) at 150 rpm during
18–20 h. The unsaponifiable material was extracted with diethyl
ether. A fraction (1/2) of the unsaponifiable material was purified by
SPE. The acetone fraction obtained was then subjected to derivatization
by silylation, using the method described for PS