MASS-SPECTROMETRIC ANALYSIS OF 4 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 prostagla ndin F2 alpha, enzymatically produced by prostaglandin H-2 synthase. I n theory, four distinct regioisomeric families are possible. These reg ioisomeric families have a common 1,3-diol cyclopentane structural fea ture, but differ by the comparative length of two attached alkyl chain s and the position of a third hydroxyl group. Eight synthetic PGF(2 al pha) isomers were found separable by capillary gas chromatography (GC) and reversed-phase high-performance liquid chromatography (HPLC). Ele ctrospray ionization tandem mass spectrometry was used to detect the e lution of these isomers from the HPLC column by monitoring the charact eristic loss of 44 u (C2H4O) from the 1,3-diol cyclopentane ring. Cata lytic reduction, derivatization, and electron ionization mass spectrom etric 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 ac id was used to generate a complex mixture of F-2-isoprostanes, which w ere separated by HPLC and capillary GC. Members of each of the four sp ecific 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 p roducts, but the complexity of the isomers required more than a simple CC-mass spectrometry assay to precisely identify a particular stereoi somer within a regioisomeric family (e.g., 8-epi-PGF(2 alpha)). Type I F-2-isoprostanes are unique noncyclooxygenase products and may be mor e specific targets to measure lipid peroxidation in vivo.