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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2017-03, Vol.1046, p.48-57
• Main Authors: Teixeira, Dulce, Prudêncio, Cristina, Vieira, Mónica
• Format: Article
• Language: English
• Subjects: 3-Nitrotyrosine; Animals; Biomarkers - analysis; Cell Line, Tumor; Chromatography, High Pressure Liquid - methods; HPLC-DAD; Humans; Limit of Detection; Linear Models; Mice; Nitrosative stress; Quantification methods; Reproducibility of Results; Tyrosine - analogs & derivatives; Tyrosine - analysis
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2017.01.035

•Increased protein nitration is associated with physiological conditions.•3-NT has been proposed for therapeutic monitoring of pathologies with nitrosative stress.•The method was successfully applied to 3-NT quantification in all biological matrices tested. The nitration of tyrosine residues in proteins is associated with nitrosative stress, resulting in the formation of 3-nitrotyrosine (3-NT).113-NT − 3-nitrotyrosine; ICH − International Conference on Harmonisation; ROS − reactive oxygen species; Try − tyrosine; RNS − reactive-nitrogen species; PYCC − Portuguese Yeast Culture Collection; MEM − Minimal Essential Medium; TSA − Trypticase Soy Agar; YEPD − yeast extract peptone dextrose. 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)-based 3-NT quantification method. All experiments were performed on an Hitachi LaChrom Elite® 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. 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 356nm, at 25°C, and using a flow rate of 1mLmin−1. By using this protocol, it was possible to obtain a linear calibration curve, limits of detection and quantification in the order of μgL−1, and a short analysis time (