62] High-pressure liquid chromatography of underivatized fatty acids, hydroxy acids, and prostanoids having different chain lengths and double-bond positions

Abstract

Thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC) methods are available that permit separation and direct visualization of submicrogram-to-milligram quantities of underivatized prostaglandins and lipoxygenase products. The TLC procedure separates primarily on the basis of number of double bonds and hydrophilicity and is not affected by small differences in hydrophobicity. In contrast, the HPLC procedure separates fatty acids and metabolites that differ only in carbon number or in the number, position, or geometry of double bonds. HPLC may also be used to resolve and quantitate underivatized fatty acids obtained from natural mixtures, such as brain glycerophospholipids or hepatic triglycerides. HPLC is simple and highly reproducible, and the same column may be used for at least 400 runs with no evidence of deterioration. It is found that baseline separations of all major oxygenated metabolites formed from arachidonic acid in platelets are readily achieved using a single ODS column. HPLC can be used to separate prostanoids of varying chain lengths. Adrenic acid is a naturally occurring fatty acid produced by chain elongation of arachidonic acid. Thus adrenic acid is arachidonic acid with two methylene groups interposed at the carboxyl(a) end. Primary prostaglandins produced from adrenic acid can be separated from those produced from arachidonic acid with one exception: TxB 2 generated from arachidonic acid overlaps the prostacyclin metabolite obtained from adrenic acid. HPLC can also be used to resolve structures with very subtle differences in hydrophobicity, e.g., 19- or 21-carbon homologs.