2.4.3. Eicosanoid Extraction and Analysis

Eicosanoid analysis was performed as previously described [35]. One hundred microliters of cord plasma and a total of 1 mg of protein from the placental homogenate were used for eicosanoid extraction. Briefly, after addition of a deuterated internal standard mixture, samples were added to MeOH (1:2), centrifuged, and then extracted using a solid phase extraction cartridge (Strata-X 33 µm Polymeric Reversed Phase, Phenomenex). Metabolites were eluted, dried down, reconstituted, injected onto an HPLC system (Shimadzu LC-10AD VP, Columbia, MD, USA) and separated on an HPLC column (Gemini C18, 150 × 2 mm, 5 µm, Phenomenex) directly interfaced into the electrospray source of a triple quadrupole mass spectrometer (5500 QTRAP, Sciex). Quantitation was performed using standard isotope dilution curves, as previously described [35].