High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

Abstract

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.