Determination of 20-hydroxyeicosatetraenoic acid in microsomal incubates using high-performance liquid chromatography–mass spectrometry (HPLC–MS)

20-HETE is a potent, vasoconstrictive arachidonic acid metabolite with a limited number of published methods for quantitative assessment of microsomal formation rate. The purpose of this study was to evaluate the utility of HPLC–MS (negative ESI) for quantitation of rat microsomal 20-HETE enzyme kinetics. Calibration curves were linear over 0.75–16 ng on-column (r2>0.996). The intra- and inter-assay precision and accuracy were <15%. Microsomal 20-HETE revealed saturable (100 μM) kinetics (brain Km and Vmax: 39.9±6.0 μM and 8.7±0.6 pmol/min per mg; liver Km and Vmax: 23.5±3.2 μM and 775.5±39.8 pmol/min per mg; kidney Km and Vmax: 47.6±8.5 μM and 1933±151 pmol/min per mg). This paper demonstrates HPLC–MS as an efficient method for quantitating 20-HETE enzyme kinetics in microsomes from rat tissues.