A novel mixed-mode solid phase extraction coupled with LC-MS/MS for the re-evaluation of free 3-nitrotyrosine in human plasma as an oxidative stress biomarker.

3-Nitrotyrosine (3-NT) has been widely adopted as a biomarker of oxidative stress. However, an enormous range of reported concentrations of 3-NT in biological matrices indicated an underlying methodological problem. Consequently, our understanding of tyrosine nitration in vivo and its significance as a biomarker may have been confounded. Here we report a fast, specific and sensitive LC-MS/MS method to accurately quantify free 3-NT in human plasma. For the first time, a single-step solid phase extraction of 3-NT using mixed-mode sorbent in a 96-well plate was developed after significant optimization. Complete chromatographic separation of 3-NT from other tyrosine analogs was achieved on a PFPP column (150 mm, 2.1 mm, 3 μm) with a cycle time of 10 min. The developed method was validated in terms of linearity, sensitivity, specificity, precision, accuracy, matrix effect, carryover, analyte stability and reference interval. The lower limit of quantification of the method was 5 pg/ml for plasma 3-NT, which represents a significant sensitivity improvement over reported methods. No artifactual 3-NT formation was observed, and the assay was not affected by 40 likely interferences. The average intra- and inter-assay variances were 3.4% and 3.7%, respectively. Of note, the reference interval for a healthy population was established to be 2.0-40.1 pg/ml (8.8-177 pM) with a mean of 11.1 pg/ml (49 pM). The mean value is at least 13-fold lower than previously reported mean values. Furthermore, the method was applied to the investigation of biological variation (n=15) over a three week period and no statistical differences were found. The solid phase extraction in a 96-well format and a fast LC-MS/MS delivered a practical, precise and accurate tool well-suited to quantify free plasma 3-NT in a large number of biological samples.