Determination of EETs using microbore liquid chromatography with fluorescence detection.
Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid involved in the regulation of vascular tone. The method of microbore column high-performance liquid chromatography with fluorescence detection 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. EETs 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 excited at 259 nm, and the fluorescence was detected at 395 nm. The overall recoveries were 88% for EETs and 40% for TA. EETs were detected in concentrations as low as 2 pg (signal-to-noise ratio = 3). The method was used to determine the EET production from endothelial cells (ECs). Bradykinin and methacholine (10(-6) M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for determination of EETs at low concentrations normally detected in complex biological samples.
Nithipatikom
K
K
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Pratt
P F
PF
Campbell
W B
WB
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003331:Coronary Vessels; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D016210:Methacholine Chloride / Q000494:pharmacology; D008832:Microchemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
D016210:Methacholine Chloride; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; C040776:5,6-epoxy-8,11,14-eicosatrienoic acid; C050715:8,9-epoxyeicosatrienoic acid; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D015126:8,11,14-Eicosatrienoic Acid; D001920:Bradykinin