Eicosanoid Analysis by LC-MS/MS

Eicosanoids were analyzed as detailed by the Lipid Maps Consortium^{33 (link),34 (link)}. Culture media (4 ml) from siRNA was combined with 10% methanol (400 μl) and glacial acetic acid (20 μl) before spiking with internal standard (100 μl) containing the following deuterated eicosanoids (100 pg/μl, 10 ng total): (d₄) 6keto-PGF₁α, (d₄) PGF₂α, (d₄) PGE₂, (d₄) PGD₂, (d₈) 5-hydroxyeicosa-tetranoic acid (5HETE), (d₈) 15-hydroxyeicosatetranoic acid (15HETE), (d₈) 14,15 epoxyeicosa-trienoic acid and (d₈) arachidonic acid. Samples and vial rinses (5% MeOH; 2 ml) were applied to Strata-X SPE columns (Phenomenex), previously washed with methanol (2 ml) and then dH₂O (2 ml). Eicosanoids eluted with isopropanol (2 ml), were dried in vacuuo and reconstituted in EtOH:dH₂O (50:50;100 μl) prior to HPLC ESI-MS/MS analysis (see Supplementary Methods).