Facile Preparation and Structural Determination of Monohydroxy Derivatives of Docosahexaenoic Acid (HDoHE) by α-Tocopherol-Directed Autoxidation

Abstract

Polyunsaturated fatty acids are oxidized through both enzymatic and nonenzymatic reactions into hydroxy derivatives. With increasing interest in dietary manipulations through ingestion of the highly unsaturated fish oil fatty acids, eicosapentaenoic acid and docosahexaenoic acid (DHA), methods to measure their metabolism are required. In this study we report the simple and expedient α-tocopherol-directed autoxidative preparation of a series of monohydroxy derivatives of DHA to provide a relatively homogenous hydroxylation along each of the double bonds of the fatty substrate. Products were purified by high-performance liquid chromatography (HPLC) and their structures elucidated by the characteristic fragmentation pattern of the hydrogenated methyl ester trimethylsilyl ether derivatives by gas chromatography-mass spectrometry. Nine products were isolated in 20.2% yield overall, ranging from 1.55 to 4.14% yield of isolated compound. These were identified as 7, 8, 10, 11, 13, 14, 16, 17, and 20-HDoHEs (monohydroxydocosahexaenoic acids). Two of these products (14- and 17-HDoHE) could not be separated under the HPLC conditions used but were clearly distinguished using selected ion chromatography by their distinct mass spectral fragmentation. This method is highly suitable for the generation of standards to investigate the metabolism of DMA in tissues.