An improved method for determining the composition of FFA in red tide flagellates by RP-HPLC with fluorescence detection

An improved method for determining the composition of FFA in red tide flagellates by HPLC with fluorescence detection is described. For this purpose, total lipids from Heterosigma akashiwo, Chattonella antiqua, and C. marina were allowed to react with 9-anthryldiazomethane, then the resulting 9-anthrylmethyl esters of FFA were analyzed without any purification by RP-HPLC on a highly efficient C18 column (Superspher 100 RP-18e, 25 cm x 4 mm i.d., 4 mum particle size; Merck, Darmstadt, Germany). Clear separations of long-chain saturated and unsaturated FFA, including 14:0 and 16:1, which were major components in the flagellates and were unresolved on a previously used C18 column, were achieved by a stepwise gradient elution using acetonitrile, water, and propan-2-ol. Two characteristic FA, 18:5n-3 and trans-16:1n-13, whose behaviors on RP-HPLC had not been reported previously, were also clearly separated from the other FFA. The FFA compositions of the flagellates determined by HPLC were in good agreement with those obtained by GLC. The present method is simple and sensitive, and would be widely applicable for compositional analysis of microalgal FFA.