Add time:07/15/2019         Source:sciencedirect.com

Publisher SummaryThe chapter presents a study on quantitation of epoxy- and dihydroxyeicosatrienoic acids by stable isotope-dilution mass spectrometry. The chapter describes a method for measurement of picomolar amounts of epoxyeicosatrienoic acids (EET) and dihydroxyeicosatrienoic acids (DHET) compounds by gas chromatography-mass spectrometry (GC-MS). The method involves preparation of 2H/3H-labeled standards of the four regionally isomeric EET compounds and the four corresponding DHET from 2H/3Hlabeled arachidonate. These labeled compounds are then used as internal standards in an assay, which involves their addition in organic solvent to terminate cell incubations, extraction, high-performance liquid chromatographic (HPLC) separation of the regionally isomeric standards (located by virtue of their 3H content), conversion to the pentafluorobenzyl ester (PFBE) derivatives (EET) or to the trimethylsilyl (TMS)-PFBE derivatives (DHET), and gas chromatographic-mass spectrometric analysis in the negative-ion (methane) chemical ionization mode H with selected-ion monitoring. The endogenous EET or DHET compounds are then quantitated relative to the 2H-labeled standards. The chapter describes the measurement of production of epoxyeicosatrienoic acids and dihydroxyeicosatrienoic acids by rat liver microsomes. The measurement is done with stable isotope-dilution gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NCI-MS).

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