High-performance liquid chromatography—thermospray mass spectrometry of hydroxy-polyunsaturated fatty acid acetyl derivatives

Abstract

A method for the determination of hydroxy-polyenoic acids has been developed. Hydroxy groups of hydroxy-polyunsaturated fatty acid were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C 18 cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, fifteen kinds of hydroxy-polyunsaturated fatty acid derivative could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatograph without a gradient system. Generally, the base ion, [M + H − n(60)] +, is produced through elimination of acetic acid ( n = number of the hydroxy group of hydroxy-polyunsaturated fatty acid). The detection limit for these derivatives was ca. 0.2 pmol at the levels of hydroxy-polyenoic acids prior to derivatization. They could be analysed in the range 0.5–25 pmol. The assay was successfully applied to hydroxy-polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acid was added.