Ice-cold methanol containing 500pg each of deuterated (d) internalstandards (d8-5S- hydroxyeicosatetraenoic, d4-LT B4, d5-LXA4, d4-PGE2,and d5-RvD2) was added to samples. Spiking with these internal lipid mediator(LM) standards enables quantification and sample recovery assessment. LM extractionand profiling were conducted as described previously (REF). In brief, LM were extracted usingsolid-phase extraction techniques and an Extra-Hera (Biotage) autoextractor (REF).
Next, liquidchromatography–tandem mass spectrometry–based LM profiling was used to identifyand quantify LM. Multiple reaction monitoring was conducted using signature Q1(parent ion) and Q3 (characteristic daughter ion) ion pairs for each molecule, acquiredin negative ionization mode. LM were identified according to published criteria,including matching retention times and with a minimum of 6 diagnostic ions inthe tandem mass spectrometry spectra (REF).