title: Development of a new HPLC-based method for 3-nitrotyrosine quantification in different biological matrices.
abstract: BACKGROUND
  The nitration of tyrosine residues in proteins is associated with nitrosative stress, resulting in the formation of 3-nitrotyrosine (3-NT).13-NT levels in biological samples have been associated with numerous physiological and pathological conditions. Hence several attempts have been made in order to develop methods that accurately quantify 3-NT in these matrices. The aim of this study was to develop a simple, rapid, low-cost and sensitive high-performance liquid chromatography (HPLC)-based 3-NT quantification method.
  
  
  METHODS
  All experiments were performed on an Hitachi LaChrom Elite®HPLC system. The method was validated according to International Conference on Harmonisation (ICH) guidelines for serum samples. Additionally, other biological matrices were tested, namely whole blood, urine, B16 F-10 melanoma cell line, growth medium conditioned with the same cell line, bacterial and yeast suspensions.
  
  
  RESULTS
  From all the protocols tested, the best results were obtained using 0.5% CH3COOH:MeOH:H2O (15:15:70) as mobile phase, with detection at wavelengths 215, 276 and 356nm, at 25°C, and using a flow rate of 1mLmin-1. By using this protocol, it was possible to obtain a linear calibration curve, limits of detection and quantification in the order of μgL-1, and a short analysis time (<15min per sample). The developed protocol allowed the successful detection and quantification of 3-NT in all biological matrices tested, with detection at 356nm.
  
  
  CONCLUSIONS
  This method, successfully developed and validated for 3-NT quantification, is simple, cheap and fast. These features render this method a suitable option for analysis of a wide range of biological matrices, being a promising useful tool for both research and diagnosis activities.
authors.affiliation: Ciências Químicas e das Biomoléculas, Centro de Investigação em Saúde e Ambiente, Escola Superior de Saúde do Porto, Instituto Politécnico do Porto, Portugal.
authors.forename: Dulce
authors.initials: D
authors.lastname: Teixeira
authors.affiliation: Ciências Químicas e das Biomoléculas, Centro de Investigação em Saúde e Ambiente, Escola Superior de Saúde do Porto, Instituto Politécnico do Porto, Portugal; I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.
authors.forename: Cristina
authors.initials: C
authors.lastname: Prudêncio
authors.affiliation: Ciências Químicas e das Biomoléculas, Centro de Investigação em Saúde e Ambiente, Escola Superior de Saúde do Porto, Instituto Politécnico do Porto, Portugal; I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal. Electronic address: mav@ess.ipp.pt.
authors.forename: Mónica
authors.initials: M
authors.lastname: Vieira
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D051379:Mice; D015203:Reproducibility of Results; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D015415:Biomarkers; C002744:3-nitrotyrosine; D014443:Tyrosine
keywords: 3-Nitrotyrosine; HPLC-DAD; Nitrosative stress; Quantification methods