Ice-cold signature Q1 (parent ion) and Q3 (characteristic daughter

Ice-cold methanol containing 500pg each of deuterated (d) internal
standards (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 extraction
and profiling were conducted as described previously (REF). In brief, LM were extracted using
solid-phase extraction techniques and an Extra-Hera (Biotage) autoextractor (REF). Next, liquid
chromatography–tandem mass spectrometry–based LM profiling was used to identify
and quantify LM. Multiple reaction monitoring was conducted using signature Q1
(parent ion) and Q3 (characteristic daughter ion) ion pairs for each molecule, acquired
in negative ionization mode. LM were identified according to published criteria,
including matching retention times and with a minimum of 6 diagnostic ions in
the tandem mass spectrometry spectra (REF).