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

Abstract

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF 2␣-F2-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 chromatography͞tandem mass spectrometry (LC͞MS͞MS), 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 2␣-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 GC͞MS and LC͞MS͞MS of iPF 2␣-VI in hypercholesterolemia and of 8,12-iso-iPF 2␣-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.

HPLC–Atmospheric Pressure Chemical Ionization MS/MS for Quantification of 15-F2t-Isoprostane in Human Urine and Plasma

“…The automated column-switching technique and on-line sample extraction reduced manual sample preparation to a simple protein precipitation step, eliminating the need for laborious sample purification and avoiding potential sources of analytical imprecision in existing methods (3,(23)(24)(25)(26)(27)32 ).…”

“…To date, SPE has been used for sample preparation in all published LC-MS methods for the analysis of 15-F 2t -IsoP (3,(23)(24)(25)(26)(27). Only one method employed automated SPE, but the extraction was carried out offline with an automated SPE workstation (27 ).…”

Intestinal and renal handling of oxalate loads in normal individuals and stone formers

“…Creatinine was measured in all urine samples as previously described [6]. Urinary isoprostanes were measured by LC/MS/ MS, as described by Li et al [8]. The urinary excretion of N-acetyl-β-glucosaminidase (NAG) and γ-glutamyl transpeptidase (GGT) was measured as previously described [9] except that urinary inhibitors of enzyme activity were removed by centrifugal filtration using Ultra-free-MC filters (Millipore Corp., Bedford, MA, USA) with a 10,000 nominal molecular weight limit.…”

F2-isoprostanes are correlated with trans fatty acids in the plasma of pregnant women

“…F 2 -isoPs must be freed from phospholipids by the action of phospholipases A 2 (PLA 2 ) in order to induce vasoconstriction in blood vessels [30,31]. The concomitant assessment of several F 2 -isoPs can provide information on the balance between generation and elimination according to the physiological state or the disorder studied [32][33][34][35].…”