Objectives: Increased lipid peroxidation (i.e. "oxidative stress") has been identified as a central mechanism in the development of atherosclerosis and inflammatory vascular damage. Measurement of 8-iso-PGF2α has demonstrated to be a reliable indicator of in vivo oxidative stress levels. The purpose of this study was to develop a rapid, sensitive, and specific LC-MS/MS method for detection of urinary 8-iso-PGF2α, establish reference intervals, and correlate isoprostane levels with cardiac troponin I. Design and methods: Urinary 8-iso-PGF2α was detected after direct injection onto a C18 silica column and monitored in the MRM mode using m/z transitions of 353.2 > 193.25 (8-iso-PGF2α) and 357.2 > 197.25 (8-iso-PGF2α-d4). The LC-MS/MS method was also compared to an ELISA kit. Reference interval studies were evaluated against a separate population of patients presenting with chest pain that had positive cTnI values. Results: Elution of 8-iso-PGF2α was achieved after 7 min, with a total run time of 10 min. Inter-assay CVs were 13.8-20.0% and intra-assay CVs were 10.9-17.0%. Linearity ranged from 100 pg/mL to 100 ng/mL. Deming regression of ELISA and LC-MS/MS methods for 8-iso-PGF2α levels yielded poor correlation, with a slope of 0.0265, y-intercept of 0.255 ng/mL, and R2 value of 0.0434. Urine 8-iso-PGF2α concentrations in samples obtained from healthy individuals (n = 34) ranged from 57 to 390 ng/g creatinine with a mean of 221 ng/g creatinine. 8-iso-PGF2α levels were statistically significant in troponin-positive (n = 35) versus troponin-negative (n = 36) patients (p < 0.0049). Conclusions: This LC-MS/MS method provides a rapid, accurate, sensitive, and cost-effective alternative to other methods for detection of 8-iso-PGF2α in urine. 8-iso-PGF2α has potential to be a great prognostic risk indicator in individuals with a high probability for future coronary events.
- Liquid chromatography-tandem mass spectrometry
- Oxidative stress