Mass spectrometric analysis of four regioisomers of F-2-isoprostanes formed by free radical oxidation of arachidonic acid

F-2-isoprostanes are complex metabolites of arachidonic acid generated via nonenzymatic free radical oxidation and are isomeric to prostaglandin F2 alpha, enzymatically produced by prostaglandin H-2 synthase. In theory, four distinct regioisomeric families are possible. These regioisomeric families have a common 1,3-diol cyclopentane structural feature, but differ by the comparative length of two attached alkyl chains and the position of a third hydroxyl group. Eight synthetic PGF(2 alpha) isomers were found separable by capillary gas chromatography (GC) and reversed-phase high-performance liquid chromatography (HPLC). Electrospray ionization tandem mass spectrometry was used to detect the elution of these isomers from the HPLC column by monitoring the characteristic loss of 44 u (C2H4O) from the 1,3-diol cyclopentane ring. Catalytic reduction, derivatization, and electron ionization mass spectrometric techniques were used to obtain definitive information as to the location of the side chain hydroxyl position in these isomers through abundant alpha-cleavage ions. Free radical oxidation of arachidonic acid was used to generate a complex mixture of F-2-isoprostanes, which were separated by HPLC and capillary GC. Members of each of the four specific regioisomeric isoprostane families could be identified in this mixture from the predicted alpha-cleavage ions. Although many epimers within a single family type could be separated, the four regioisomeric families were substantially superimposed in their HPLC and GC elution. The Type I and Type TV regioisomers were the major F-2-isoprostane products, but the complexity of the isomers required more than a simple CC-mass spectrometry assay to precisely identify a particular stereoisomer within a regioisomeric family (e.g., 8-epi-PGF(2 alpha)). Type I F-2-isoprostanes are unique noncyclooxygenase products and may be more specific targets to measure lipid peroxidation in vivo.