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A simple and robust LC‐MS/MS method for quantification of free 3‐nitrotyrosine in human plasma from patients receiving on‐pump CABG surgery
Author(s) -
Hui Yu,
Wong Michael,
Zhao Shuai Sherry,
Love Jennifer A.,
Ansley David M.,
Chen David D.Y.
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100368
Subject(s) - detection limit , chromatography , mass spectrometry , chemistry , analyte , nitrotyrosine , human plasma , cardiopulmonary bypass , solid phase extraction , medicine , anesthesia , nitric oxide , nitric oxide synthase , organic chemistry
We have developed a simple, sensitive, and robust liquid chromatography‐mass spectrometry/mass spectrometry (LC‐MS/MS) method to determine free 3‐nitrotyrosine concentrations in human plasma of patients receiving on‐pump coronary artery bypass grafting surgery. A one‐step solid‐phase extraction protocol was optimized to enrich the analyte at low nanomolar concentrations. The processed samples were analyzed by LC‐MS/MS with a 2.1 × 100 mm Kinetex PFP column and a triple quadrupole mass spectrometer. The method was validated for 3‐nitrotyrosine concentrations close to real patient plasma levels. The relative standard deviations or relative errors of the intraday and interday determinations were all within 10%. Limit of detection and limit of quantitation were determined to be 0.034 nM and 0.112 nM, respectively, while lower limit of quantitation was below 0.625 nM. No deterioration of the column performance was noticed after running a large number of patient samples. The results showed that the 3‐nitrotyrosine concentrations in coronary sinus plasma samples were elevated after cardiopulmonary bypass (CPB) procedure. The pre‐CPB and post‐CPB concentrations of 3‐nitrotyrosine in patient plasmas were 1.494 ± 0.107 nM and 2.167 ± 0.177 nM (mean ± SEM), respectively. Application of this method to more patients in clinical studies may help validate 3‐nitrotyrosine as a meaningful biomarker for nitrosative stress and link patient characteristics, clinical outcomes, and cardioprotective treatments to endogenous nitrosative stress levels.