Analysis of cytochrome P450 metabolites of arachidonic and linoleic acids by liquid chromatography mass spectrometry with ion trap MS2

We have used reversed phase-high performance liquid chromatography-mass spe ctrometry (RP-HPLC-MS) with an ion trap mass spectrometer to study the meta bolism of arachidonic and linoleic acids by human recombinant cytochrome P4 50 (CYP) enzymes. We first recorded the MS2 spectra of the carboxylate anio ns of epoxides, diols, omega-side chain, and bisallylic hydroxy fatty acids of arachidonic, octadeuterated arachidonic, and linoleic acids. The metabo lites formed by CYP2C9 and CYP2C19 were then studied. CYP2C9 converted arac hidonic and linoleic acids to epoxides/diols and monohydroxy fatty acids. S ome hydroxyeicosatetraenoic acids (HETEs) were studied in detail to investi gate the oxygenation mechanism. Incubation of CYP2C9 under oxygen-18 gas sh owed that all HETEs had incorporated oxygen-is to the same degree. Chiral H PLC showed that CYP2C9 formed 15R-HETE (72% of the R enantiomer), 13S-HETE (90%), and 11R-HETE (57%). RP-HPLC-MS analysis revealed that CYP2C19 oxygen ated arachidonic acid to 19-HETE, 14,15-epoxyeicosatrienoic acid (EET), and 8,9-EET as main metabolites. The method was sufficiently sensitive to iden tify arachidonic acid metabolites formed by some other isozymes. RP-HPLC-MS with MS2 seems to be useful for rapid identification of fatty acid metabol ites in complex mixtures formed by cytochrome P450. (C) 1998 Academic Press .