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

Abstract

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF_2α—F₂-iPs—in urine has reflected lipid peroxidation in humans. However, up to 64 F₂-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 F₂-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF_2α-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 F₂-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC/MS and LC/MS/MS of iPF_2α-VI in hypercholesterolemia and of 8,12-iso-iPF_2α-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.