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A Sensitive LC-MS/MS Method for the Quantification of Regioisomers of Epoxyeicosatrienoic and Dihydroxyeicosatrienoic Acids in Human Plasma During Endothelial Stimulation

A Sensitive LC-MS/MS Method for the Quantification of Regioisomers of Epoxyeicosatrienoic and Dihydroxyeicosatrienoic Acids in Human Plasma During Endothelial Stimulation

Thomas Duflot, Tony Pereira, Clothilde Roche, Michèle Iacob, Pascal Cardinael, Najla El-Gharbi Hamza, Christian Thuillez, Patricia Compagnon, Robinson Joannidès, Fabien Lamoureux, Jérémy Bellien

Anal Bioanal Chem. 2017 Mar;409(7):1845-1855.

PMID: 27981341

Abstract:

Epoxyeicosatrienoic acids (EETs) are vasodilating lipid mediators metabolized into dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase. We aimed to develop a LC-MS/MS method to quantify EETs and DHETs in human plasma and monitored their levels during vascular endothelial stimulation. Plasma samples, collected from 14 healthy and five hypertensive subjects at baseline and during radial artery endothelium-dependent flow-mediated dilatation, were spiked with internal standards. Lipids were then extracted by a modified Bligh and Dyer method and saponified to release bound EETs and DHETs. Samples were purified by a second liquid-liquid extraction and analyzed by LC-MS/MS. The assay allowed identification of (±)8(9)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-EET); (±)11(12)-epoxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-EET); (±)14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-EET); (±)8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-DHET); (±)11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-DHET); and (±)14,15-dihydroxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-DHET). (±)5(6)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (5,6-EET) was virtually undetectable due to its chemical instability. The limits of quantification were 0.25 ng/mL for DHETs and 0.5 ng/mL for EETs. Intra- and inter-assay variations ranged from 1.6 to 13.2%. Heating induced a similar increase in 8,9-EET, 11,12-EET, and 14,15-EET levels and in corresponding DHET levels in healthy but not in hypertensive subjects. We validated a sensitive LC-MS/MS method for measuring simultaneously plasma EET and DHET regioisomers in human plasma and showed its interest for assessing endothelial function.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP123931408	11,12-Epoxy-(5Z,8Z,14Z)-eicosatrienoic acid		123931-40-8	Price
AP69254371	Arachidonic-5,6,8,9,11,12,14,15-d8 acid		69254-37-1	Price

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