Fatty acids in erythrocytes measured by isocratic HPLC

We developed an isocratic reversed-phase HPLC method to measure arachidonic, palmitoleic, linoleic, eicosatrienoic, oleic, palmitic, and stearic acids from hydrolyzed erythrocytes. Washed erythrocytes were heated in methanol:HCl and the fatty acids extracted into hexane:amyl alcohol. After derivatization with 4-bromomethyl-7-methoxycoumarin, samples diluted in mobile phase (acetonitrile:water, 85:15 by vol) were injected onto a 250 x 4.6 mm C18 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 mumol/L, a linear response was maintained up to 400 mumol/L, and results agreed well with those by gas chromatography. The addition of antioxidant (butylated hydroxytoluene) was essential for sample stability. We discuss hydrolysis and extraction times, derivatization temperature, critical steps in chromatography, and concentration units.