Quantitative high performance liquid chromatography tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF(2 alpha)-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 F2-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F2-iPs, using liquid chromatography/tandem mass spectrometry (LC/MS/MS), in which sample preparation is minimized. Authentic standards of F-2-iPs of classes Ill, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using IPF2 alpha-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F2-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 IPF2 alpha-VI in hypercholesterolemia and of 8,12-iso-iPF(2 alpha)-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.