Targeted Lipid Mediator Profiling

All samples for LC-MS-MS-based profiling were extracted using solid-phase extraction columns (9). Three microliter of peritoneal exudate were placed in ice-cold methanol containing deuterated internal standards, representing each region in the chromatographic analysis (500 pg each). Samples were kept at -20°C for 45 min to allow protein precipitation. Supernatants were subjected to solid phase extraction, methyl formate fraction collected, brought to dryness and suspended in phase (methanol/water, 1:1, vol/vol) for injection on a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector, paired with a QTrap 5500 (Sciex). An Agilent Poroshell 120 EC-C18 column (100 mm × 4.6 mm × 2.7 μm) was kept at 50°C and mediators eluted using a mobile phase consisting of methanolwater-acetic acid of 20:80:0.01 (vol/vol/vol) that was ramped to 50:50:0.01 (vol/vol/vol) over 0.5 min and then to 80:20:0.01 (vol/vol/vol) from 2 min to 11 min, maintained till 14.5 min and then rapidly ramped to 98:2:0.01 (vol/vol/vol) for the next 0.1 min. This was subsequently maintained at 98:2:0.01 (vol/vol/vol) for 5.4 min, and the flow rate was maintained at 0.5 ml/min. The QTrap 5500 was operated using a multiple reaction monitoring method. Each LM was identified using established criteria including matching retention time to synthetic and authentic materials and at least six diagnostic ions (9).