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Determination of plasma free 3‐nitrotyrosine and tyrosine by reversed‐phase liquid chromatography with 4‐fluoro‐7‐nitrobenzofurazan derivatization
Author(s) -
Zhang WeiZheng,
Lang Charles,
Kaye David M.
Publication year - 2007
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.750
Subject(s) - chemistry , chromatography , derivatization , tyrosine , reversed phase chromatography , high performance liquid chromatography , biochemistry
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 n m 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 n m and 1–320 µ m , 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 n m and 0.263 ± 0.192 n m /µ m with 3.8 and 3.9 times higher (both p < 0.05), respectively, than that of non‐smoker controls (4.54 ± 2.75 n m and 0.067 ± 0.050 n m /µ m , 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. Copyright © 2007 John Wiley & Sons, Ltd.