FAST HPLC DETERMINATION OF SERUM-FREE FATTY-ACIDS IN THE PICOMOLE RANGE

We developed a method for determining individual free fatty acids in s erum by using a modified one-step Dole extraction, derivatization, and a new high-performance liquid chromatographic (HPLC) separation. Samp le handling is minimized to a single transfer of the fatty acids (uppe r layer of the Dole extract), which are readily derivatized at 85-degr ees-C with p-bromophenacyl bromide without significant hydrolysis of e sterified fatty acids. The derivatization mixture is directly injected into the HPLC apparatus. The new method, which uses C6 (3-mum particl e) column material and an isocratic acetonitrile-water eluent, separat es nearly to baseline 12 of the physiologically most abundant long-cha in fatty acids (C-12-C22) in <20 min with a detection limit of approxi mately 2 pmol. It is therefore suitable for routine analysis even with basic HPLC equipment and can easily analyze a series of 10-20 samples in about 2 h including extraction until first results are available. The method is also applicable to other matrices than serum, e.g., for determination of precursor fatty acids such as arachidonic acid in pla telets or of fatty acid patterns liberated by lipases or phospholipase s A1/A2 in test systems.