Liquid chromatography/electrospray ionisation mass spectrometric tracking of 4-hydroxy-2(E)-nonenal biotransformations by mouse colon epithelial cells using [1,2-13C2]-4-hydroxy-2(E)-nonenal as stable isotope tracer

4-Hydroxy-2(E)-nonenal (HNE), a product of lipid peroxidation, has been extensively studied in several areas, including metabolism with radio-isotopes and quantification in various matrices with deuterium-labelled HNE as standard. The aim of this work was to evaluate the relevance of C-13-labelled HNE in biotransformation studies to discriminate metabolites from endogens by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS). C-13-Labelled HNE was synthesised in improved overall yield (20%), with the incorporation of two labels in the molecule. Immortalised mouse colon epithelial cells were incubated with 2: 3 molar amounts of HNE/C-13-HNE in order to gain information on the detection of metabolites in complex media. Our results demonstrated that the stable isotope m/z values determined by mass spectrometry were relevant in distinguishing metabolites from endogens, and that metabolite structures could be deduced. Six conjugate metabolites and 4-hydroxy-2(E)-nonenoic acid were identified, together with an incompletely identified metabolite. Stable-isotope-labelled HNE has already been used for quantification purposes. However, this is the first report on the use of C-13-labelled HNE as a tracer for in vitro metabolism. C-13-Labelled HNE could also be of benefit for in vivo studies. Copyright (C) 2011 John Wiley & Sons, Ltd.