Determination of Total Long-Chain Fatty Acids in Human Plasma and Lipoproteins, before and during Copper-Stimulated Oxidation, by High-Performance Liquid Chromatography

Abstract

An improved method for the high-performance liquid chromatographic determination (HPLC) of total or free long-chain (> C12) fatty acids in small volumes (10 microL) of human plasma and lipoprotein samples is described. The method is based on the formation of 2-nitrophenyl-hydrazine (2-NPH) derivatives and offers an alternative to gas chromatographic (GC) fatty acid determination. The retention of 2-NPH fatty acid derivatives on the HPLC system differs from the typical pattern produced by GC separation, thus offering a powerful tool for confirmation of peak identification where GC peak resolution is poor. Fatty acids determined include saturates [myristic acid, C14:0; palmitic acid, C16:0; stearic acid, C18:0; eicosanoic acid, C20:0; docosanoic acid, C22:0; and tetracosanoic acid, C24:0], monounsaturates [palmitoleic, C16:1; petroselenic, C18:1n12; oleic, C18:1n9; and erucic, C22:1], and polyunsaturates [linoleic, C18:2; linolenic, C18:3n3; gamma-linolenic acid, C18:3n6; eicosatrienoic, C20:3; arachidonic, C20:4; eicosapentanoic, C20:5; docosahexanoic, C20:6; and docosatetraenoic, C22:4]. Mean recoveries of fatty acids added to LDL samples were 94.1-109.4%, and intraassay coefficients of variation for the major fatty acids in human plasma were 2.7-6.9%. The potential of the method for further development is discussed. Long-chain fatty acid profiles are given for plasma and very low-, low, and high-density lipoprotein (before and during copper-stimulated oxidation) from human blood.