Determination of Isoprostanes as Marker Oxidant Stress by LC-ESI/MS

Abstract

The isoprostane, 8-iso-PGF2_α is formed from arachidonic acid in vivo by a mechanism independent of cyclooxygenase pathway. We developed a new assay method for 8-iso-PGF2_α using [²H₄]-8-iso-PGF2_α as the internal standard (I.S.) by LC-ESI/MS. For this assay, we established a very simple and rapidly pretreatment method using a membrane filter-type solid phase extraction column (Empore™ disk cartridge) for human plasma and urine. LC-ESI/MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 353.4757 (8-iso-PGF2_α) and m/z 357.5073 (I.S.) with a resolution of 3,000. The imprecision for this method was below 14%. Mean inaccuracy was 9% for added levels of 8-iso-PGF2_α up to 5,000 pg/mL of urine and 500 pg/mL of plasma. The study of human urinary and plasma 8-iso-PGF2_α concentrations may be a convenient diagnostic tool to be able to assess the extent of oxidative stress in vivo not only by smoking but also in other disease states.