as an IS and CH3CN (10L). Then, 30L perchloric acid (60%)
was added for deproteinization, and 800L chloroform, 100L nheptane,
and 100L H2O were added for the extraction of fatty
acids. After the solution was centrifuged at 3000×g for 5 min, the
organic layer (400L) was transferred to a tube and evaporated
under reduced pressure without heating for 22 min. Following the
addition of 10mMp-toluenesulfonic acid (PTSA) in MeOH (200L)
to the dried residue and incubation at 62 ◦C for 2 h for methyl
esterification, H2O (200L) and n-hexane (200L) were sequentially
added, mixed for 2min, and centrifuged at 4000×g for 5min.
The hexane layer (150L), which included fatty acid methyl esters
(FAMEs), was obtained. Then, 10L sample solution was subjected
to GC–MS analysis.