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Updated: Sep 20, 2026
Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
Published on: November 17, 2011
Determination of 20-hydroxyeicosatetraenoic acid in microsomal incubates using high-performance liquid
Christopher A Bolcato1, Reginald F Frye, Michael A Zemaitis
1Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Abstract:
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 (r(2)>0.996). The intra- and inter-assay precision and accuracy were <15%. Microsomal 20-HETE revealed saturable (100 microM) kinetics (brain K(m) and V(max): 39.9+/-6.0 microM and 8.7+/-0.6 pM/min per mg; liver K(m) and V(max): 23.5+/-3.2 microM and 775.5+/-39.8 pmol/min per mg; kidney K(m) and V(max): 47.6+/-8.5 microM and 1933+/-151 pM/min per mg). This paper demonstrates HPLC-MS as an efficient method for quantitating 20-HETE enzyme kinetics in microsomes from rat tissues.
