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 deuteriumlabeled 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.Key words epoxyeicosatrienoic acid; dihydroxyeicosatrienoic acid; arachidonic acid; LC-MS/MS Arachidonic acid (AA) is metabolized to various eicosanoids via several pathways.1) These eicosanoids play important roles in physiological processes, including the regulation of inflammation.2-5) Three major metabolic pathways for conversion of AA to eicosanoids are recognized, including routes that incorporate cyclooxygenase (COX), lipoxygenase (LOX), and CYP.6) The CYP pathway, which is mediated primarily by CYP2C9, CYP2C8, and CYP2J2, produces epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs). 7,8) EETs exist as one of 4 regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET), depending on the location of the epoxidized residue. Each EET is substantially metabolized to its corresponding diol, a dihydroxyeicosatrienoic acid (DHET), by soluble epoxide hydrolase (sEH).1,9-12) The EETs have been shown to play important roles on cardiovascular homeostasis. [13][14][15] Thus, the drug inhibition of CYP-mediated AA metabolism via could reduce production of EETs, potentially resulting adverse cardiovascular events.Traditional analytical methods such as HPLC or capillary electrophoresis with UV or fluorescence detection, 16,17) and GC-MS 18) lack sufficient sensitivity to detect the low levels of AA metabolites typically found in physiological matrices. Sev...

Angiotensin II Receptor Blockers Inhibit the Generation of Epoxyeicosatrienoic Acid from Arachidonic Acid in Recombinant CYP2C9, CYP2J2 and Human Liver Microsomes

“…Eicosanoids were determined by LC‐MS/MS as described in detail by Mukai et al . . All incubations were within the linear range of the AA metabolism with respect to the incubation duration and P450 content.…”

“…Incubation was performed using the method of Mukai et al . with some modifications. Each ARB stock solution was evaporated and reconstructed to create the final concentration (0, 10, 50, 100 and 500 μM) in the incubation medium.…”

Effects of Angiotensin II Receptor Blockers on Metabolism of Arachidonic Acid <i>via</i> CYP2C8

“…18) An Agilent 1200 series (Santa Clara, CA, U.S.A.) LC system was used to separate samples. Separation was performed on an Ascentis Express C18 column measuring 100 mm ×2.1 mm with a particle size of 2.7 µm (Supelco, Sigma-Aldrich, St. Louis, MO, U.S.A.).…”

The Inhibitory Effect of Telmisartan on the Metabolism of Arachidonic Acid by CYP2C9 and CYP2C8: An <i>in Vitro</i> Study

“…20,21) An Agilent 1200 series HPLC system (Santa Clara, CA, U.S.A.) was used with a binary pump, a degasser, and a thermostatic autosampler. HPLC separation was achieved using an Ascentis Express C18 column (10 cm×2.1 mm, 2.7 µm, Sigma-Aldrich).…”

“…We previously developed a method for the simultaneous quantification of EETs and DHETs by LC-MS/MS. 20) Using this method, we demonstrated that several ARBs inhibit AA metabolism in recombinant CYP (rCYP) 2C8, 21) rCYP2C9, rCYP2J2, and HLMs 22) ; among the ARBs, telmisartan exhibited more potent inhibitory effect towards the CYP enzymes. 21,22) Several studies have revealed that telmisartan inhibits CYP2C9 23) and CYP2J2 24) in HLMs.…”