Determination of 5-hydroperoxyeicosatetraenoic acid produced in rat basophilic leukemia cell line RBL-2H3 by high-performance liquid chromatography with chemiluminescence detection.

A simple and sensitive method, applicable to quantification of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) produced in cells has been developed using high-performance liquid chromatography on a silica gel column with chemiluminescence detection. 5-HPETE was clearly separated from other positional isomers of HPETEs and hydroxyeicosatetraenoic acids with hexane-isopropanol-acetic acid (97:3:0.01, v/v) as the mobile phase. The lower limit of detection was about 100 pg. 5-HPETE produced in 10(7) cells of RBL-2H3 cells stimulated with A23187 was determined as 480+/-30 pg. In the present study, 5-HPETE, which occurs naturally, was detected and quantitated for the first time in intact cells.