The LC-MS-MS system, QTrap 5500 (ABSciex), was equipped with an Agilent HP1100 binary pump and diode-array detector (DAD). An Agilent Eclipse Plus C18 column (100 mm × 4.6 mm × 1.8 μm) was used with a gradient of methanol/water/acetic acid of 60:40:0.01 (v/v/v) to 100:0:0.01 at 0.4 ml/min flow rate. To monitor and quantify the levels of the various LM, a multiple reaction monitoring (MRM) method was developed with signature ion fragments for each molecule. Identification was conducted using published criteria17 (link) with at least six diagnostic ions. Calibration curves were obtained using synthetic LM mixture (d8-5S-HETE, d4LTB4, d5LXA4, d4PGE2, TXB2, PGD2, PGF2α, RvD1, RvD2, RvD5, Protectin (PD)1, Maresin 1 (MaR1), 17-hydroxydocosahexaenoic acid (17-HDHA), 14-hydroxydocosahexaenoic acid (14-HDHA) and 7-hydroxydocosahexaenoic acid (7-HDHA) at 1, 10, 100, 275 pg. Linear calibration curves for each were obtained with r2 values in the range 0.98–0.99. Quantification was carried out based on peak area of the Multiple Reaction Monitoring (MRM) transition and the linear calibration curve for each compound. Where calibration curves for a structurally related DHA-derived product were not available (14,21-diHDPA, 13,14-diHDPA and 16,17-diHDPA), levels were monitored using a compound with similar physical properties.
For chiral lipidomic analysis, a Chiralpak AD-RH column (150 mm × 2.1 mm × 5 μm) was used with isocratic methanol/water/acetic acid 95:5:0.01 (v/v/v) at 0.15 ml/min. To monitor isobaric monohydroxy docosapentaenoic acid levels, a multiple reaction monitoring (MRM) method was developed using signature ion fragments for each molecule.