HPLC analysis of essential fatty acids in rat plasma

Linoleic acid (n-6 family) and -Linolenic acid (n-3 family) are two polyunsaturated fatty acids (PUFAs) that are known as essential fatty acids and must be provided by diet.They play an important role in coagulation and inflammatory reactions; concerning cardiovascular diseases studies demonstrated a decreased mortality in populations who eat an omega-3 rich diet or who take an omega-3 supplement.This paper deals with HPLC analysis of linoleic acid and linolenic acid in rat plasma in order to investigate the effect of flaxseed oil supplementation on plasma levels of these essential fatty acids.HPLC procedures are recommended for accurately quantifying of thermally labile moieties like polyunsaturated fatty acids; nevertheless a derivatization step is required as these analytes are lacking both in chromophore and fluorophore group and cannot therefore be detected at sufficiently low concentrations.In this work, p-bromophenacyl bromide is used as UV-derivatizing agent followed by HPLC/UV on C18 reversed phase column. The mobile phase gradient was studied in order to obtain a complete separation for the anaytes and the internal standard from matrix components.Concerning plasma sample clean-up, existing techniques of lipid extraction were tested and modified to achieve maximal free fatty acids recovery in a reasonable time.Studies on rats fed with flaxseed oil are currently in progress in order to evaluate how the chronic supplementation can increase plasma levels of n-3 and n-6 fatty acids.