Quantitative analysis of 5-oxo-6,8,11,14-eicosatetraenoic acid by electrospray mass spectrometry using a deuterium-labeled internal standard

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a metabolite of arachido nic acid formed by the 5-lipoxygenase pathway, is a potent eosinophil chemo attractant that may be an important mediator in asthma. To further investig ate the physiological and pathological roles of 5-oxo-ETE we have developed a mass spectrometric assay employing a tetra-deuterated analog (5-oxo-[11, 12,14,15-H-2]ETE) as an internal standard. Collision-induced dissociation o f the quasimolecular anion of 5-oxo-[11,12,14,15-H-2]ETE (m/z 321) resulted in the formation of a major ion at m/z 207 that retained all four deuteriu m atoms. Measurement of the ratio of ions at m/z 203 (endogenous 5-oxo-ETE) and m/z 207 permitted quantitation of this compound by liquid chromatograp hy-mass spectrometry-mass spectrometry using multiple reaction monitoring. The resulting assay was highly sensitive (less than or equal to 20 pg/sampl e) and selective, enabling detection of the amount of 5-oxo-ETE produced by as few as 10,000 neutrophils. This assay should permit measurement of 5-ox o-ETE in biological fluids, enabling evaluation of its role in asthma and o ther inflammatory diseases. (C) 2001 Academic Press.