3-Nitrotyrosine quantification methods: Current concepts and future challenges

Measurement 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.