GC–MSanalysiswasperformed usingGCcoupled to amass spectrometer
(QP2010; SHIMADZU, Japan). Separation of the FAMEs
was performed on a polar capillary column (DB-23; J&W Scientific,
Folsom, UK) with 60m×0.25mm×0.25m film thickness. The
carrier gas was helium. The temperature program was optimized
to separate each FAME quickly as follows: initial temperature 50 ◦C,
held for 2min, increased at 10 ◦C/min to 200 ◦C, and kept at 200 ◦C
for 10 min. Finally, the temperature was increased at 10 ◦C/min to
220 ◦C for another 15 min. The mass spectrometer was operated in
the electron impact ionizationmodeat 70 eV. Both the interface and
ionization source were kept at 230 ◦C. The purchased FAME mixture,
which was diluted 100 times with n-hexane, was analyzed
first. Subsequently, based on their order of elution and the retention
times, each fatty acid was confirmed without decomposition.
The fatty acids in serum sample were determined by their retention
times and them/z values of their molecular ions by comparison
with the chromatograms and mass spectra analyzed beforehand.
Measurements were then performed in the SIM mode using the
two most intense fragment ions for quantification and confirmation
(Table 1). Stock solutions of the nine fatty acids and heptadecanoic
acid (IS) were prepared at 30 and 15mM in CH3CN:DMF (1:1) and
stored at –20 ◦C. Working solutions were made up with CH3CN at
concentrations ranging from 0.5 to 800M according to the concentration
of each fatty acid in the serum samples. The peak area
ratio of the IS was then plotted against each concentration (n = 4).