Determination of 8-epi PGF(2alpha) concentrations as a biomarker of oxidative stress using triple-stage liquid chromatography/tandem mass spectrometry.

F2-isoprostanes are a family of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid. Several F2-isoprostanes, but in particular 8-epi PGF(2alpha), are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8-epi PGF(2alpha) concentrations in human plasma, whole blood, erythrocytes and urine. 8-epi PGF(2alpha)-d4, a stable isotope derivative of 8-epi PGF(2alpha), was used as an internal standard (IS). A 50 microL sample was focused on-column and separated on two 3 microm particle size SUPELCOSIL ABZ+Plus HPLC columns (15 cm x 4.6 mm and 7.5 cm x 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 353.4 --> 193.1 (8-epi PGF(2alpha)), 357.4 --> 197.1 (8-epi PGF(2alpha)-d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples.