Liquid chromatographic-electrospray ionization-mass spectrometric analysisof cytochrome P450 metabolites of arachidonic acid

Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes t o many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,1 5-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrie noic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These ei cosanoids are potent regulators of vascular tone. We developed a liquid chr omatography-electrospray ionization-mass spectrometry method to simultaneou sly determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 1 4,15-DHETs; and 20-HETE. [H-2(8)]EETs, [H-2(8)]DHETs, and [H-2(2)]20-HETE w ere used as internal standards. These compounds are readily separated on a C-18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [H-2(8)]EETs, [H-2(8)]DHETs, and [H-2(2)]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and d etected in a negative mode. The negative ion detection gave a lower backgro und than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1](-). The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-H ETE, respectively. The detection limits using selected ion monitoring of th ese compounds are about 1 pg per injection. The position of functional grou ps and water content of mobile phase had a significant effect on the sensit ivity of detection. Water content of 40% was found to give maximal sensitiv ity. The method was used to determine EETs, DHETs, and 20-HETE in bovine co ronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples. (C) 2001 Academic Press.