Simultaneous determination of 3-nitrotyrosine and tyrosine in plasma proteins of rats and assessment of artifactual tyrosine nitration

A sensitive and specific isotope dilution liquid chromatography-electrospray tandem mass spectrometry method was developed for the determination of the 3-nitrotyrosine residue levels in rat plasma proteins. The assay is based on the cleavage of proteins with concentrated hydrochloric acid to release both 3-nitrotyrosine and tyrosine. To control the potential artifactual nitration of tyrosine residues during the proteolysis, samples are spiked with ¹³C₉-labeled tyrosine and the level of ¹³C₉-labeled 3-nitrotyrosine is measured. The clean-up process entails hydrolysate fortification with 2,5,6-d₃-3-nitrotyrosine, followed by solid-phase extraction on octadecylsilyl (to isolate tyrosine) and aminopropylsilyl (to isolate 3-nitrotyrosine) cartridges. Tyrosine and 3-nitrotyrosine fractions are mixed in an appropriate ratio prior to the analysis. The method was applied to animals exposed to ferric nitrilotriacetate to induce oxidative stress.