LC-MS/MS methods for the detection of isoprostanes (iPF2alpha-III and 8,12-iso-iPF2alpha-VI) as biomarkers of CCl4-induced oxidative damage to hepatic tissue.

Isoprostanes are formed after peroxidation of arachidonic acid and are promising biomarkers for reactive oxygen species. A LC-MS/MS based method was developed for the quantitation of two isoprostanes (iPF2alpha-III and 8,12-iso-iPF2alpha-VI) in hepatocytes, tissue and urine samples of rats. A column switching method was used to reduce sample preparation to a minimum. Precision was 9.4% and accuracy was between 96 and 114% for free iPF2alpha-III in tissue at concentrations from 1.9 to 6.1 ng/g. Treatment of rats with CCl4 to induce oxidative stress resulted in a dose-dependent increase (two- to three-fold) of iPF2alpha-III and 8,12-iso-iPF2alpha-VI in liver and kidney. For both isoprostanes an increase of four- to five-fold was observed in CCl4 treated hepatocytes and six- to eight-fold in CCl4 treated and glutathione depleted hepatocytes. In conclusion, the presented method is sensitive, specific and precise to be applied for the quantitation of iPF2alpha-III and 8,12-iso-iPF2alpha-VI which are shown to increase by CCl4 treatment in vitro and in vivo.