Distinct macrophage subtypes biosynthesize specific lipid mediator profiles. M1 and M2 macrophages were prepared from primary human monocytes after incubation with GM-CSF (20 ng/mL), IFN-γ (20 ng/mL), and LPS (100 ng/mL) to produce M1 or M-CSF (20 ng/mL) and IL-4 (20 ng/mL) to obtain M2. Incubations were stopped with ice-cold MeOH and taken for LC-MS/MS analysis (see “Sample extraction and lipid mediator metabololipidomics”). (A) Representative MRM chromatograms for the identified LM. Peak heights represent the relative levels of each mediator in the different macrophage subtypes. Cumulative levels for each lipid mediator category are represented as a function of color intensity, where color scales (eg, white to green for M1 macrophages and white to purple for M2 macrophages) are set from zero to 35 000 pg per 2.5 × 10⁶ cells. (B) Accompanying MS/MS spectra used for identification. (C) Lipid mediator and precursor/pathway marker transition along with mean ± SEM values for each of the mediators identified. The detection limit was ∼ 1 pg. *Below limits. Cumulative values: (D) D-series resolvins, protectins, and maresins. (E) Lipoxins. (F) E-series resolvins and lipoxins. (G) Prostaglandins and thromboxanes. (H) EPA-derived prostaglandins and thromboxanes. (I) Leukotriene B₄ (and 20-OH LTB₄ metabolite). (J) Leukotriene B₅. (D-J) Results are ± SEM; n = 8 distinct cell preparations. *P < .05 vs M1 group. **P < .01 vs M1 group.