Determination of EETs using microbore liquid chromatography with fluorescence detection

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachi donic acid involved in the regulation of vascular tone. The method of micro bore column high-performance liquid chromatography with fluorescence detect ion was developed to determine 14,15-EET, 11,12-EET, and the mixture of 8,9 -EET and 5,6-EET. Tridecanoic acid (TA) was used as an internal standard. E ETs were reacted with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (NT) to form highly fluorescent derivatives. A C-18 microbore column and a water-acetonitrile mobile phase were used for separation. Samples were exci ted at 259 nm, and the fluorescence was detected at 395 nm. The overall rec overies were 88% for EETs and 40% for TA. EETs were detected in concentrati ons as low as 2 pg (signal-to-noise ratio = 3). The method was used to dete rmine the EET production from endothelial cells (ECs). Bradykinin and metha choline (10(-6) M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for dete rmination of EETs at low concentrations normally detected in complex biolog ical samples.