Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography

• Published in: Journal of chromatography. B, Biomedical applications Vol. 685; no. 2; pp. 343 - 347
• Main Authors: Kamisaki, Yoshinori, Wada, Kouichirou, Nakamoto, Kentaro, Kishimoto, Yosuke, Kitano, Masayuki, Itoh, Tadao
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 25.10.1996; Elsevier
• Subjects: 4-Chloro-7-nitrobenzofurazan - analogs & derivatives; 4-Chloro-7-nitrobenzofurazan - chemistry; Biological and medical sciences; Biomarkers - blood; Cell metabolism, cell oxidation; Cell physiology; Chromatography, High Pressure Liquid - methods; Fluorescent Dyes - chemistry; Fundamental and applied biological sciences. Psychology; Humans; Molecular and cellular biology; Nitrotyrosine; Reproducibility of Results; Sensitivity and Specificity; Tyrosine - analogs & derivatives; Tyrosine - blood; Tyrosine - chemistry; Nitrotyrosine; Human; Biological fluid; Oxidative stress; Fluorescence detector; Investigation method; Reaction product; HPLC chromatography; Oxidant; Isocratic condition; Quantitative analysis; Blood plasma
• ISSN: 0378-4347; 1572-6495
• DOI: 10.1016/S0378-4347(96)00202-2

A highly sensitive and simple isocratic high-performance liquid chromatography method was developed for determination of 3-nitrotyrosine in human plasma with precolumn derivatization with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole. The precision of the method was satisfactory (coefficient of variation 4.8%), and the detection limit was established at 0.1 pmol of 3-nitrotyrosine allowing the determination at the level of 6 pmol/ml in human plasma. The recoveries of 3-nitrotyrosine and α-methyltyrosine, an internal standard, were 89.3 +-7.1 and 85.7±7.6%, respectively. The 3-nitrotyrosine level was 31±6 pmol/ml (mean±S.D., n=9) in plasma from healthy volunteers. Since 3-nitrotyrosine is a stable product of peroxynitrite, an oxidant formed by a reaction of nitric oxide and superoxide radicals, the measurement of its plasma concentration may be useful as a marker of nitric oxide-dependent oxidative damage.