It is understood that exposure to free radicals related to tobacco use negatively impacts smoker health. Studies have been performed measuring fatty acid oxidation products (F2-Isoprostanes) in human urine as biomarkers of harm from free radical exposure. A challenge noted with this approach is that the measured biomarkers are formed through enzymatic pathways and chemical lipid peroxidation initiated by free radical exposure. A novel approach to differentiate the biomarker components specific to the enzymatic and radical peroxidation pathways was developed by van’t Erve et al. (Free Radical Biology and Medicine 2015). The analysis of a ratio of stereo-isomers, 8-iso-prostaglandin F_2α (8-iso-PGF_2α), and prostaglandin F_2α (PGF_2α) in human plasma was suggested as a possible method improvement in the determination of oxidative stress. Initial published concentration results from non-smoker subjects ranged from approximately 45 to 110 pg/mL for both analytes. The mean ratio non-smoker plasma samples of 8-iso-PGF_2α and PGF_2α was reported as 0.88 ± 0.26.

Working to further investigate this biomarker of oxidative stress, an assay was developed to measure the stereo-isomers in human plasma samples. To aid in reproducibility of the measurement, the samples were stabilized with indomethacin (inhibition of cyclooxygenase-1 and -2) and butylated hydroxytoluene (BHT) (inhibition of chemical peroxidation). A selective analytical approach was developed with reversed phase gradient chromatography on a UPLC system prior to detection by MS/MS on a SCIEX Triple Quad^® 6500. Samples were prepared for injection via mixed-mode solid phase extraction.

Samples collected with the added inhibitors were significantly lower in measured concentrations of both analytes with a range of 6 to 15 pg/mL. Mean ratios of 8-iso-PGF_2α and PGF_2α observed in smoker and non-smoker samples were between 0.77-1.14 and 0.95-1.12 pg/mL respectively. Further evaluation of the ratios was performed with samples incubated in an attempt to reproduce the published work.