FATTY-ACIDS IN ERYTHROCYTES MEASURED BY ISOCRATIC HPLC

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.