LC/MS/MS method for analysis of E₂ series prostaglandins and isoprostanes

15-series prostaglandins (PGE₂s) and isoprostanes (isoPGE₂s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE₂/D₂) and isoPGE₂ without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE₂/PGD₂/isoPGE₂ such as PGE₂, entPGE₂, 8-isoPGE₂, 11β-PGE₂, PGD₂, and 15(R)-PGD₂ to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE₂ in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE₂ levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE₂s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.