Details of the manipulation and purification of the
samples have been published before [13,14]. Briefly, the
most superficial layers of the samples were cut with a
bladder and discarded, and accurately weighed parts of
tissues were homogenized with anhydrous sodium sulfate. After the addition of internal standards (free FAs
12:0 for dolphins and 13:0 for turtles) and an acidic
solution of methanol (1 N H2
SO4
), the headspace of the
tubes were filled with pure N2
, the content mixed with
a vortex and the tubes placed in an oven at 60–80°C
for several hours, shaking the tubes occasionally.
FAMEs were extracted with n-hexane after adding
distilled water. Optimal conditions of transmethylation
of the FAs and reproducibility tests were checked and
performed during the development of the method
[13,14].