Enzymatically Formed Fatty Acid Hydroperoxides Determined Through GC-MS Analysis of Enantiomeric Excess of Hydroxy Fatty Acids After Reduction and Ibuprofen Derivatization

Abstract

Unsaturated fatty acids are susceptible to lipid oxidation through autoxidation, photooxygenation or enzymatical oxidation. A characteristic feature of enzyme-catalyzed oxidation is the high regio- and stereospecificity of the formed fatty acid hydroperoxides. In this study, we present a method to quantify enzymatic lipid oxidation through reducing hydroperoxy fatty acid methyl esters to hydroxy fatty acid methyl esters and derivatizing them with enantiopure (S)-ibuprofen, allowing the resolution of the enantiomer pairs as diastereomers via achiral GC-MS. After application to enantiopure reference fatty acids, the approach was applied to autoxidation products of linoleic acid, and the expected racemic mixtures of the 9- and 13-hydroperoxide derived hydroxy fatty acids were detected. On the other hand, when linoleic acid was oxidized using soybean lipoxygenase, clear enantiomeric excess of the (13S) enantiomer could be detected, proving the applicability of this method to detect enzymatic oxidation through enantiomeric excess.