Rapid simultaneous analysis of cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic and linoleic acids using high performance liquid chromatography/mass spectrometry in tandem mode

Abstract

Eicosanoids are oxidized arachidonate-derived lipid products generated by cyclooxygenase, lipoxygenase and cytochrome P-450 pathways. They are involved in diverse processes in health and disease and they are highly bioactive. Gas chromatography and enzyme immunoassays were used to quantify these mediators in the past. However, the recent availability of high-sensitivity liquid chromatography–mass spectrometry has provided a new approach for quantification that minimizes the sample size and the required preparation. This paper describes a rapid and simple technique for the simultaneous quantitative analysis of prostaglandin (PG) E 2 and PGJ 2; leukotrienes (LT) B 4 and D 4; 5-, 12-, 15- and 20-hydroxyeicosatetraenoic acids (HETEs); 13-hydroxyoctadecadienoic acid (13-HODE); 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs); and 11,12- and 14,15-dihydroxieicosatrienoic acids (DHETs) in cell culture supernatants and urine. We simultaneously analyzed 14 arachidonic acid metabolites representative from the three pathways, together with 13-HODE, a linoleic-derived product. Solid phase extraction was used for the sample preparation. The recoveries obtained ranged from 25% to 100%, depending on the metabolites. The LC/MS/MS method used the gradient on a C 18 column and electrospray ionization in negative ion detection mode. The method was optimized for sensitivity and for separation within 20 min. The linear ranges of the calibration curves were 0.1–200 ng/ml for PGE 2, PGJ 2, LTB 4, 5-HETE, 12-HETE, 15-HETE, 13-HODE, 11,12-EET, 11,12-DHET and 14,15-DHET, and 1–200 ng/ml for LTD 4, 20-HETE, 5,6-EET, 8,9-EET and 14,15-EET. The advantages of this method include minimal sample preparation, high sensitivity and elimination of the problem associated with thermal instability in gas chromatography analysis.