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Oxidative Medicine and Cellular Longevity
Volume 2016, Article ID 1746985, 7 pages
Research Article

Validation of a Reversed-Phase High Performance Liquid Chromatography Method for the Simultaneous Analysis of Cysteine and Reduced Glutathione in Mouse Organs

1Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61029 Urbino, Italy
2Department of Public Health and Infectious Diseases, Pasteur Institute, Cenci-Bolognetti Foundation, “Sapienza” University of Rome, 00161 Rome, Italy
3San Raffaele Pisana Scientific Institute for Research, Hospitalization, and Health Care, 00163 Rome, Italy

Received 4 August 2015; Revised 5 October 2015; Accepted 7 October 2015

Academic Editor: Jara Perez-Jimenez

Copyright © 2016 Serena Brundu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


A depletion of reduced glutathione (GSH) has been observed in pathological conditions and in aging. Measuring GSH in tissues using mouse models is an excellent way to assess GSH depletion and the potential therapeutic efficacy of drugs used to maintain and/or restore cellular redox potential. A high performance liquid chromatography (HPLC) method for the simultaneous determination of GSH and cysteine (Cys) in mouse organs was validated according to USA and European standards. The method was based on separation coupled with ultraviolet detection and precolumn derivatization with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The required validation parameters, that are, selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, and stability, were studied for spleen, lymph nodes, pancreas, and brain. The results showed that the lower limits of quantification were 0.313 μM and 1.25 μM for Cys and GSH, respectively. Intraday and interday precisions were less than 11% and 14%, respectively, for both compounds. The mean extraction recoveries of Cys and GSH from all organs were more than 93% and 86%, respectively. Moreover, the stability of both analytes during sample preparation and storage was demonstrated. The method was accurate, reliable, consistent, and reproducible and it was useful to determine Cys and GSH in the organs of different mouse strains.