LC-MS/MS Determination of Isoprostanes in Plasma Samples Collected from Mice Exposed to Doxorubicin or Tert-Butyl Hydroperoxide

Isoprostanes are stable products of arachidonic acid peroxidation and are regarded as the most reliable markers of oxidative stress in vivo. Here we describe the LC-MS/MS procedure enabling simultaneous determination of four regioisomers (8-iso prostaglandin F(2α), 8-iso-15(R)-prostaglandin F(2α), 11β-prostaglandin F(2α), 15(R)-prostaglandin F(2α)) in plasma samples collected from mice. The four plasma isoprostanes are determined by LC–ESI-MS/MS with deuterated 8-iso-PGF(2α)-d(4) as an internal standard (I.S.). For plasma samples spiked with the isoprostanes at a level of 200 pg/mL each, the method imprecision has been below 7.1% and mean inaccuracy equaled 8.7%. The applicability of the proposed approach has been verified by the assessment of changes in isoprostane levels in plasma samples derived from mice exposed to tert-butyl hydroperoxide (TBHP), a model inducer of oxidative stress, or to antitumor drug doxorubicin (DOX) known for potent stimulation of redox cycling. Compared to the control group of mice, both oxidative stress inducers tested increased the levels of three out of four isoprostanes in exposed animals; 11β-prostaglandin F(2α) being the exception. The greatest rise was observed in the case of 15(R)-prostaglandin F(2α), by about 50% and 70% in plasma samples derived from mice exposed to DOX and TBHP, respectively.