We developed an isocratic reversed-phase HPLC method to measure arachi
donic, palmitoleic, linoleic, eicosatrienoic, oleic, palmitic, and ste
aric acids from hydrolyzed erythrocytes. Washed erythrocytes were heat
ed in methanol:HCl and the fatty acids extracted into hexane:amyl alco
hol. After derivatization with 4-bromomethyl-7-methoxycoumarin, sample
s diluted in mobile phase (acetonitrile:water, 85:15 by vol) were inje
cted onto a 250 x 4.6 mm C-18 column, and the eluted fatty acids were
detected fluorometrically. For all analytes, the mean within-batch CV
was 8.2% (5.5-10.8%), the mean limit of detection was 7.0 mu mol/L, a
linear response was maintained up to 400 mu mol/L, and results agreed
well with those by gas chromatography. The addition of antioxidant (bu
tylated hydroxytoluene) was essential for sample stability. We discuss
hydrolysis and extraction times, derivatization temperature, critical
steps in chromatography, and concentration units.