This chapter presents the highly sensitive and selective assay for 5-hydroxyeicosatetraenoic acid (5-HETE) employing racemic octadeuterated 5-HETE for stable isotope dilution measurements with quantitation via combined gas-liquid chromatography-mass spectrometry (GC–MS). Measurement of 5-HETE is employed as an index of 5-1ipoxygenation of arachidonic acid. Octadeuterated arachidonic acid is prepared from 5,8,11,14-eicosatetraynoic acid. Two independent methods are employed for standardization of ds-5- HETE, because the tendency of 5-HETE to form δ-lactone. Ultraviolet spectrophotometry at, λMeOHmax = 235 nm and gas–liquid chromatography with tricosanoic acid as internal standard should give agreement within ±5%. In purification by reversed-phase high performance liquid chromatography (HPLC), solvent is evaporated from the sample extract under a stream of nitrogen, and the residue is dissolved in a small volume of methanol for purification by reversed-phase HPLC. 5-HETE is eluted from μBondapak C18 column with a solvent of methanol-water -glacial acetic acid delivered at a flow rate of 1-2 ml/min.
ASJC Scopus subject areas
- Molecular Biology