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Determination of Plasma Free 3-nitrotyrosine and Tyrosine by Reversed-Phase Liquid Chromatography With 4-fluoro-7-nitrobenzofurazan Derivatization

Determination of Plasma Free 3-nitrotyrosine and Tyrosine by Reversed-Phase Liquid Chromatography With 4-fluoro-7-nitrobenzofurazan Derivatization

Wei-Zheng Zhang, Charles Lang, David M Kaye

Biomed Chromatogr. 2007 Mar;21(3):273-8.

PMID: 17236239

Abstract:

Oxidative stress plays an important role in pathogenesis of many diseases. Measurement of 3-nitrotyrosine (NO(2)Tyr), as a potential biomarker for nitric oxide-mediated damage, has recently been the focus of particular attention. We have developed an HPLC method with NBD-F pre-column derivatization followed by C(18) cartridge cleaning. Using this method we achieved limits of detection of 0.5 and 1.1 nm for NO(2)Tyr and tyrosine (Tyr), respectively, close to that achieved by LS-MS/MS. NO(2)Tyr and tyrosine concentrations were linear over the calibration ranges 0.5-100 nm and 1-320 microm, respectively, with correlation coefficients greater than 0.95. To evaluate the utility of this assay in plasma we analysed samples obtained from smokers and non-smoking subjects. Consistent with the presence of elevated oxidative stress, the plasma NO(2)Tyr concentration and NO(2)Tyr:Tyr ratio of smokers were 17.42 +/- 11.6 nm and 0.263 +/- 0.192 nm/microm with 3.8 and 3.9 times higher (both p < 0.05), respectively, than that of non-smoker controls (4.54 +/- 2.75 nm and 0.067 +/- 0.050 nm/microm, respectively). In conclusion, we have developed a novel HPLC assay for NO(2)Tyr without MS detection that is applicable to clinical studies addressing the pathophysiology and importance of oxidative stress.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP29270562	4-Fluoro-7-nitrobenzofurazan		29270-56-2	Price

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