Isoprostanes (IsoPs) are key biomarkers for investigating the role of free radical generation in the pathogenesis of human disorders. To solve IsoPs-related problems with regard to isoprostanes, analytical tools are required. This paper reviews the problems and trends in this field focusing on the methodology for assaying biomarkers in exhaled breath condensate (EBC) samples. A large amount of work has been done in the qualitative and quantitative analysis of IsoPs, but a standardized method has yet to emerge. The methodologies described differ, either in the sample preparation steps or in the detection techniques, or both. Requiring a number of chromatographic steps, the relevant extraction and purification procedures are often critical and time-consuming, and they lead to a substantial loss of target compounds. Recent data show that EBC is a promising non‑invasive tool for the evaluation of different diseases. Two main analytical approaches have been adopted for IsoPs measurement: immunological methods and mass spectrometry. The methodologies for the extraction, purification and analysis of IsoPs in EBC samples are presented.Isoprostanes (IsoPs) are key biomarkers for investigating the role of free radical generation in the pathogenesis of human disorders. To solve IsoPs-related problems with regard to isoprostanes, analytical tools are required. This paper reviews the problems and trends in this field focusing on the methodology for assaying biomarkers in exhaled breath condensate (EBC) samples. A large amount of work has been done in the qualitative and quantitative analysis of IsoPs, but a standardized method has yet to emerge. The methodologies described differ, either in the sample preparation steps or in the detection techniques, or both. Requiring a number of chromatographic steps, the relevant extraction and purification procedures are often critical and time-consuming, and they lead to a substantial loss of target compounds. Recent data show that EBC is a promising non‑invasive tool for the evaluation of different diseases. Two main analytical approaches have been adopted for IsoPs measurement: immunological methods and mass spectrometry. The methodologies for the extraction, purification and analysis of IsoPs in EBC samples are presented.

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-d₄ 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.