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)-based 3-NT quantification method.

Methods: 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.

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 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 (<15min per sample). The developed protocol allowed the successful detection and quantification of 3-NT in all biological matrices tested, with detection at 356nm.

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.

• PMID: 28131027
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Journal Article

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Teixeira D, Prudêncio C, Vieira M

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