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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

作者:Xiaoguang Sunny, Li;Shu, Li;Gottfried, Kellermann

来源:Talanta 2015 年 140卷

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作者:
Xiaoguang Sunny, Li;Shu, Li;Gottfried, Kellermann
来源:
Talanta 2015 年 140卷
标签:
3-Nitrotyrosine LC–MS/MS Method validation Mixed-mode solid phase extraction Oxidative stress biomarker Plasma
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