Quantifying Skin Eicosanoids by LC-MS/MS

Eicosanoids in skin blister fluid were quantified by liquid chromatography coupled to electrospray ionization tandem mass spectrometry as described previously (30 (link)–32 (link)). Briefly, skin fluid samples (typically 50–200 μL) were diluted with methanol-water (15% w/w) up to 3 mL. Internal standards (40 ng PGB₂-d4 and 80 ng 12-HETE-d8; Cayman Chemicals) were then added and resultant solutions acidified to pH 3.0, followed by solid-phase extraction (C18-E cartridges; Phenomenex) to reduce matrix effects and semi-purify the lipid mediators. Eicosanoids were analyzed on a C18 column (Luna 5 μm; Phenomenex) by using a Waters Alliance 2695 HPLC pump coupled to a triple-quadrupole mass spectrometer equipped with an electrospray ionization probe (Quattro Ultima; Waters). Instrument control and data acquisition were performed by using MassLynx 4.0 software (Waters). Multiple-reaction monitoring transitions used were as follows: PGE₂, m/z 351 > 271; PGE₁, m/z 353 > 317; PGE₃, m/z 349 > 269; PGJ₂, m/z 333 > 271; PGD₁, m/z 353 > 317; PGD₂, m/z 351 > 271; PGF_2α, m/z 353 > 193; 13,14-dihydro-15-keto PGE₂, m/z 351 > 333; 13,14-dihydro-15-keto-PGE₁, m/z 353 > 335; 12-HETE, m/z 319 > 179; 11-HETE, m/z 319 > 167; 15-HETE, m/z 319 > 175; 15-hydroxyeicosatrienoic acid, m/z 321 > 221; 9-hydroxyoctadecadienoic acid, m/z 295 > 171; and 13-hydroxyoctadecadienoic acid, m/z 295 > 195.