Samples were kept
on ice under nitrogen during work up and were stored
at 2208C under argon. Total lipid was saponified with
200 mL of 0.1 M KOH in 95% (by vol.) ethanol under
argon for 1 h at 788C. Five hundred microlitre of water
was added and non-saponifiable material removed by
extracting with 500 mL of isohexane/diethyl ether (2:1,
by vol.), the aqueous phase acidified with 40 mL of 6M
HCl and the resulting free fatty acids extracted with
2 500 mL diethyl ether. Pentafluorobenzyl esters were
then prepared from one-third of the sample using acetonitrile/
diisopropylamine/pentafluorobenzyl bromide
(1000:10:1, v/v) at 608C for 30 min under nitrogen as
described by Pawlosky et al., (Pawlosky et al., 1992).
Excess reagent and solvent were removed under
oxygen-free nitrogen; samples were dissolved in isohexane
and stored at 2208C under argon until analysis.