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- Development of a new HPLC-based method for 3-nitrotyrosine quantification in different biological matricesPublication . Teixeira, Dulce; Prudêncio, Cristina; Vieira, MónicaThe 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 356 nm, at 25 °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 μg 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.
- 3-Nitrotyrosine quantification methods: Current concepts and future challengesPublication . Teixeira, Dulce; Fernandes, Rúben; Prudêncio, Cristina; Vieira, MónicaMeasurement of 3-nitrotyrosine (3-NT) in biological samples can be used as a biomarker of nitrosative stress, since it is very stable and suitable for analysis. Increased 3-NT levels in biological samples have been associated with several physiological and pathological conditions. Different methods have been described for the detection and quantification of this molecule, such as (i) immunological methods; (ii) liquid chromatography, namely high-pressure liquid chromatography (HPLC)-based methods that use ultraviolet-visible (UV/VIS) absorption, electrochemical (ECD) and diode array (DAD) detection, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS); (iii) gas chromatography, such as gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Methods A literature review on nitrosative stress, protein nitration, as well as 3-NT quantification methods was carried out. Results This review covers the different methods for analysis of 3-NT that have been developed during the last years as well as the latest advances in this field. Overall, all methods present positive and negative aspects, although it is clear that chromatography-based methods present good sensitivity and specificity. Regarding this, GC-based methods exhibit the highest sensibility in the quantification of 3-NT, although it requires a prior time consuming derivatization step. Conversely, HPLC does not require such derivatization step, despite being not as accurate as GC. Conclusion It becomes clear that all the methods described during this literature review, although accurate for 3-NT quantification, need to be improved regarding both sensitivity and specificity. Moreover, optimization of the protocols that have been described is clearly needed.