title: Simultaneous Determination Method of Epoxyeicosatrienoic Acids and Dihydroxyeicosatrienoic Acids by LC-MS/MS System.
abstract: Epoxyeicosatrienoic acids (EETs) are produced primarily by CYPs from arachidonic acid (AA) and then further metabolized to the corresponding dihydroxyeicosatrienoic acids (DHETs). EETs play important roles in physiological processes such as regulating vasodilation and inflammation. Thus, the drug inhibition of CYP-mediated AA metabolism could reduce production of EETs, potentially resulting in adverse cardiovascular events. The aim of this study was to develop a simple method to simultaneously determine the concentrations of both EETs and DHETs using a conventional LC-MS/MS system to evaluate drug-endogenous substance interactions, including eicosanoids. Eight eicosanoids (5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5,6-DHET, 8,9-DHET, 11,12-DHET, and 14,15-DHET) were detected with their corresponding deuterium-labeled eicosanoids as internal standards. The samples were purified by solid-phase extraction columns. Liquid chromatographic separation was achieved on a C18 column. DHETs and EETs were eluted at 4-7 and 18-26 min, respectively. The weighted (1/y(2)) calibration curves were linear over a range of 5-2000 nmol/L for EETs and 2-2000 nmol/L for DHETs. In quality control (QC) samples, the recoveries of eicosanoids were 95.2-118%. The intra-day precisions were within 6% in all three QC samples, and the inter-day precisions were <16.7% at 50 nmol/L, <8.6% at 200 nmol/L, and <9.8% at 1000 nmol/L. We have applied this method for the determination of the eicosanoid levels in samples from incubation studies of AA by using human recombinant CYP enzyme (rCYP), and confirmed that the method has sensitivity sufficient for assessment of rCYP incubation study.
authors.affiliation: Division of Clinical Pharmacology, Hokkaido Pharmaceutical University School of Pharmacy.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Mukai
authors.forename: Takaki
authors.initials: T
authors.lastname: Toda
authors.forename: Satoya
authors.initials: S
authors.lastname: Takeuchi
authors.forename: Asuna
authors.initials: A
authors.lastname: Senda
authors.forename: Miki
authors.initials: M
authors.lastname: Yamashita
authors.forename: Erik
authors.initials: E
authors.lastname: Eliasson
authors.forename: Anders
authors.initials: A
authors.lastname: Rane
authors.forename: Nobuo
authors.initials: N
authors.lastname: Inotsume
mesh_terms: D002851:Chromatography, High Pressure Liquid; D065729:Cytochrome P-450 CYP2C9 / Q000737:chemistry / Q000235:genetics; D015777:Eicosanoids / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry
chemical_list: D015777:Eicosanoids; D011994:Recombinant Proteins; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9