The extraction procedure for serum fatty acids and subsequent methyl esterification were performed according to previously published reports with minor modifications [7,19]. Ten microliters of human serum was mixed with 50M heptadecanoic acid (10L) 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.