Development of a new HPLC-based method for 3-nitrotyrosine quantification in different biological matrices

作者：Teixeira Dulce; Prudencio Cristina; Vieira Monica^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2017, 1046: 48-57.

DOI：10.1016/j.jchromb.2017.01.035

• 3-Nitrotyrosine
• Nitrosative stress
• HPLC-DAD
• Quantification methods

Background: The nitration of tyrosine residues in proteins is associated with nitrosative stress, resulting in the formation of 3-nitrotyrosine (3-NT).(1) 3-NT levels in biological samples have been associated with numerous physiological and pathological conditions. Hence several attempts have been made in order to develop methods that accurately quantify 3-NT in these matrices. The aim of this study was to develop a simple, rapid, low-cost and sensitive high-performance liquid chromatography (HPLC)-basecl 3-NT quantification method.
Methods: All experiments were performed on an Hitachi LaChrom Elite (R) HPLC system. The method was validated according to International Conference on Harmonisation (ICH) guidelines for serum samples. Additionally, other biological matrices were tested, namely whole blood, urine, B16 F-10 melanoma cell line, growth medium conditioned with the same cell line, bacterial and yeast suspensions.
Results: From all the protocols tested, the best results were obtained using 0.5% CH3COOH:MeOH:H2O (15:15:70) as mobile phase, with detection at wavelengths 215, 276 and 356 nm, at 25 degrees C, and using a flow rate of 1 mL min(-1). By using this protocol, it was possible to obtain a linear calibration curve, limits of detection and quantification in the order of mu g L-1, and a short analysis time (<15 min per sample). The developed protocol allowed the successful detection and quantification of 3-NT in all biological matrices tested, with detection at 356 nm.
Conclusion: This method, successfully developed and validated for 3-NT quantification, is simple, cheap and fast. These features render this method a suitable option for analysis of a wide range of biological matrices, being a promising useful tool for both research and diagnosis activities.

• 出版日期2017-3-1