Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F ₂ -isoprostanes in human urine

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF(2alpha)-F(2)-iPs-in urine has reflected lipid peroxidation in humans. However, up to 64 F(2)-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F(2)-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F(2)-iPs, using liquid chromatography/tandem mass spectrometry (LC/MS/MS), in which sample preparation is minimized. Authentic standards of F(2)-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF(2alpha)-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F(2)-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F(2)-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC/MS and LC/MS/MS of iPF(2alpha)-VI in hypercholesterolemia and of 8, 12-iso-iPF(2alpha)-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.