Li Xiaoguang Sunny, Li Shu, Kellermann Gottfried
Pharmasan Labs, Inc., 373 280th Street, Osceola, WI 54020, USA.
Pharmasan Labs, Inc., 373 280th Street, Osceola, WI 54020, USA.
Talanta. 2015 Aug 1;140:45-51. doi: 10.1016/j.talanta.2015.02.053. Epub 2015 Mar 6.
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.
3-硝基酪氨酸（3-NT）已被广泛用作氧化应激的生物标志物。然而，生物基质中报道的3-NT浓度范围差异巨大，这表明存在潜在的方法学问题。因此，我们对体内酪氨酸硝化作用及其作为生物标志物的意义的理解可能已受到混淆。在此，我们报告一种快速、特异且灵敏的液相色谱-串联质谱（LC-MS/MS）方法，用于准确量化人血浆中的游离3-NT。经过大量优化后，首次开发出在96孔板中使用混合模式吸附剂对3-NT进行单步固相萃取的方法。在PFPP柱（150 mm，2.1 mm，3 μm）上实现了3-NT与其他酪氨酸类似物的完全色谱分离，循环时间为10分钟。所开发的方法在线性、灵敏度、特异性、精密度、准确度、基质效应、残留、分析物稳定性和参考区间等方面进行了验证。该方法对血浆3-NT的定量下限为5 pg/ml，相较于已报道的方法，灵敏度有显著提高。未观察到人为的3-NT形成，且该测定不受40种可能干扰物的影响。批内和批间平均变异分别为3.4%和3.7%。值得注意的是，健康人群的参考区间确定为2.0 - 40.1 pg/ml（8.8 - 177 pM），平均值为11.1 pg/ml（49 pM）。该平均值至少比先前报道的平均值低13倍。此外，该方法应用于为期三周的生物变异研究（n = 15），未发现统计学差异。96孔板形式的固相萃取和快速LC-MS/MS提供了一种实用、精确且准确的工具，非常适合在大量生物样品中量化游离血浆3-NT。
