Determination of Arachidonic Acid and Its Endogenous Eicosanoid Metabolites in Mice Tissues Using Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry

A reversed phase liquid chromatography/tandem mass spectrometry method was developed to simultaneously analyze arachidonic acid(AA) and its metabolites such as prostaglandins(PGs), thromboxane (TXB(2)), epoxyeicosatrienoic acids(EETs), dihydroxyeicosatrienoic acids(DHETs) and hydroxyeicosatetraenoic acids(HETEs) through the corresponding cyclooxygenase, cytochrome P450, and lipoxygenase pathways in mice tissues. Ethyl acetate extraction was employed for sample preparation after sonication in an ice bath, and the organic phase was dried under vacuum and dissolved in methanol for the LC-MS/MS analysis. 22 standard compounds were separated within 50 min using a gradient elution of acetonitrile and water with 0.05% formic acid (V/V) on a C(18) column (250 mm x 4.6 mm, 5 mu m). PGB(1) and deuterium labeled 15-HETE-d8 and AA-d8 were used as internal standards. Mass spectrometric detection was performed using negative electrospray ionization and multiple reaction monitoring. The method was validated and shown to be accurate(recovery values 70.7% - 123.0%), precise(RSD < 20%) and sensitive(LOQ 3 - 250 mu g/L). The method was applied to separate arachidonic acid(AA) and its metabolites in lung, liver and spleen tissues in mice and quantify eicosanoid levels in lung tissue. The results were 11-HETE (58.3 +/- 11.6) ng/g, 15-HETE (52.6 +/- 15.5) ng/g, PGE(2)(210 +/- 102) ng/g, PGD(2) (20.3 +/- 13.9) ng/g, PGJ(2)(39.2 +/- 15.9) ng/g, TXB(2) (58.3 +/- 28.4) ng/g, PGF(2 alpha)(3.8 +/- 6.0) ng/g and AA(1980 +/- 544) ng/g, respectively