Rapid quantitation of free fatty acids in human plasma by high-performanceliquid chromatography

We report a rapid and sensitive method for separation and quantitation of f ree fatty acids (FFAs) in human plasma using high-performance liquid chroma tography (HPLC). Two established techniques of lipid extraction were invest igated and modified to achieve maximal FFA recovery in a reasonably short t ime period. A modified Dole extraction method exhibited greater recovery (s imilar to 90%) and short processing times (30 min) compared to the method o f Miles et al. Reversed-phase HPLC using UV detection was used for plasma F FA separation and quantitation. Two phenacyl ester derivatives, phenacyl br omide and p-bromophenacyl bromide, were investigated in order to achieve op timal separation of individual plasma FFAs (saturated and unsaturated) with desirable detection limits. Different chromatographic parameters including column temperature, column type and elution profiles (isocratic and gradie nt) were tested to achieve optimal separation and recovery of fatty acids. Phenacyl bromide esters of plasma fatty acids were best resolved using an o ctadecylsilyl column with endcapped silanol groups. An isocratic elution me thod using acetonitrile-water (83:17) at 2 ml/min with UV detection at 242 nm and a column temperature of 45 degrees C was found to optimally resolve the six major free fatty acids present in human plasma (myristic [14:0], pa lmitic [16:0], palmitoleic [16:1], stearic [18:0], oleic [18:1] and linolei c [18:2]), with a run time of less than 35 min and detection limits in the nmol range. The entire process including plasma extraction, pre-column deri vatization, and HPLC quantitation can be completed in similar to 90 min wit h plasma samples as small as 50 mu l. Over a wide physiological range, plas ma FFA concentrations determined using our HPLC method agree closely with m easurements using established TLC-GC methods (r(2) greater than or equal to 0.95). In addition, by measuring [C-14] or [H-3] radioactivity in eluent f ractions following HPLC separation of plasma FFA, this method can also quan titate rates of FFA turnover in vivo in human metabolic studies employing i sotopic tracers of one or more fatty acids. (C) 1998 Elsevier Science B.V. All rights reserved.