title: Validation of a Reversed-Phase High Performance Liquid Chromatography Method for the Simultaneous Analysis of Cysteine and Reduced Glutathione in Mouse Organs.
abstract: 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.
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", 61029 Urbino, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Brundu
authors.affiliation: Department of Public Health and Infectious Diseases, Pasteur Institute, Cenci-Bolognetti Foundation, "Sapienza" University of Rome, 00161 Rome, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Nencioni
authors.affiliation: Department of Public Health and Infectious Diseases, Pasteur Institute, Cenci-Bolognetti Foundation, "Sapienza" University of Rome, 00161 Rome, Italy.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Celestino
authors.affiliation: Department of Public Health and Infectious Diseases, Pasteur Institute, Cenci-Bolognetti Foundation, "Sapienza" University of Rome, 00161 Rome, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Coluccio
authors.affiliation: Department of Public Health and Infectious Diseases, Pasteur Institute, Cenci-Bolognetti Foundation, "Sapienza" University of Rome, 00161 Rome, Italy; San Raffaele Pisana Scientific Institute for Research, Hospitalization, and Health Care, 00163 Rome, Italy.
authors.forename: Anna Teresa
authors.initials: AT
authors.lastname: Palamara
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", 61029 Urbino, Italy.
authors.forename: Mauro
authors.identifier: 0000-0001-6456-6626
authors.initials: M
authors.lastname: Magnani
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", 61029 Urbino, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Fraternale
mesh_terms: D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D003545:Cysteine / Q000032:analysis*; D005260:Female; D005978:Glutathione / Q000032:analysis*; D057230:Limit of Detection; D008813:Mice, Inbred ICR; D009928:Organ Specificity; D015203:Reproducibility of Results
chemical_list: D005978:Glutathione; D003545:Cysteine