Improved assay for the quantification of the major urinary metabolite of the isoprostane 15-F2t-isoprostane (8-iso-PGF2α) by a stable isotope dilution mass spectrometric assay

Background: The F-2-isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical catalyzed peroxidation of arachidonic acid. One IsoP, 15-F-2t-IsoP (8-iso-PGF(2 alpha)), has been shown to be formed in abundance in vivo and to exert potent biological activity. Methods: As a means to assess the endogenous production of this compound, we previously developed a method to quantify the major urinary metabolite of 15-F-2t-IsoP, 2,3-dinor-5,6-dihydrol-15-F-2t-IsoP (2,3-dinor-5,6-dihydro-8-iso-PGF(2 alpha), 15-F-2t-IsoP-M), by gas chromotography (GC)/negative ion chemical ionization mass spectrometry (MS) employing stable isotope dilution methodology. While useful, we found that the assay occasionally suffered from the presence of impurities that co-elute on GC with 15-F-2t-IsoP-M, making the measurement of this compound difficult. We now report a modified assay for the quantification of 15-F-2t-IsoP-M employing GC/MS that alleviates this problem. Results: Precision of the assay is +/-7% and the accuracy is 96%. The lower limit of sensitivity is approximately 8 pg. Normal concentrations of this metabolite in urine were found to be 0.46 +/- 0.09 ng/mg creatinine (mean +/- 1 S.D.) Urinary excretion of 15-F-2t-IsoP-M is markedly altered in situations associated with increased or decreased oxidant stress in vivo. Conclusions: This assay provided a sensitive and accurate method to assess endogenous IsoP generation and can be used to further explore the role of oxidant injury in human disease. (C) 2001 Elsevier Science B.V. All rights reserved.