Qualitative and quantitative measurement of hydroxy fatty acids, thromboxanes and prostaglandins using stable isotope dilutions and detection by gas chromatography—mass spectrometry

Abstract

Methods for measurement of the metabolites of arachidonic acid (AA), namely prostaglandins (PGs), thromboxanes (TXs) and hydroxy fatty acids, using stable isotope dilution gas chromatography—mass spectrometry are described. With a few exceptions, labelled species of the various AA metabolites are not commercially available and were therefore synthesized in our laboratory. [ 2H 8] AA, produced by deuteration of eicosatetraynoic acid, was used for comparing the metabolism of exogenously added and endogenously present AA in fibroblast cultures. After derivatization and catalytic hydrogenation, structure elucidation and quantification of the different hydroxy fatty acids was carried out by determination of the fragment ions resulting form α-cleavage at the site of the hydroxy function. During catalytic hydrogenation a significant hydrogen—deuterium exchange was observed. To eliminate this problem, 18O-labelled standards were prepared by exchanging the oxygen of the carboxylic acid group. The preparation and the use of hydroxy fatty acids, PGs and TXs labelled with 18O is described.