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

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.

[1]  R. Sarpong,et al.  Bio-inspired synthesis of xishacorenes A, B, and C, and a new congener from fuscol† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc02572c , 2019, Chemical science.

[2]  T. Funatsu,et al.  Analytical methods involving separation techniques for determination of low-molecular-weight biothiols in human plasma and blood. , 2014, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[3]  Mingcheng Guo,et al.  Simultaneous determination of glutathione, cysteine, homocysteine, and cysteinylglycine in biological fluids by ion-pairing high-performance liquid chromatography coupled with precolumn derivatization. , 2014, Journal of agricultural and food chemistry.

[4]  A. Palamara,et al.  Molecules Altering the Intracellular Thiol Content Modulate NF-kB and STAT-1/IRF-1 Signalling Pathways and IL-12 p40 and IL-27 p28 Production in Murine Macrophages , 2013, PloS one.

[5]  R. Apak,et al.  Determination of biothiols by a novel on-line HPLC-DTNB assay with post-column detection. , 2012, Analytica chimica acta.

[6]  Q. Gao,et al.  Use of isotope differential derivatization for simultaneous determination of thiols and oxidized thiols by liquid chromatography tandem mass spectrometry. , 2011, Analytical biochemistry.

[7]  P. Ghezzi Role of glutathione in immunity and inflammation in the lung , 2011, International journal of general medicine.

[8]  Bing Li,et al.  A Highly Selective Fluorescent Probe for Detection of Biological Samples Thiol and Its Application in Living Cells , 2010, Journal of Fluorescence.

[9]  W. Tseng,et al.  Gold nanoparticle extraction followed by capillary electrophoresis to determine the total, free, and protein-bound aminothiols in plasma. , 2010, Analytical chemistry.

[10]  Hongqiao Zhang,et al.  Glutathione: overview of its protective roles, measurement, and biosynthesis. , 2009, Molecular aspects of medicine.

[11]  Shusheng Zhang,et al.  Determination of physiological thiols by electrochemical detection with piazselenole and its application in rat breast cancer cells 4T-1. , 2008, Journal of the American Chemical Society.

[12]  D. Darmaun,et al.  Simultaneous determination of glutathione and cysteine concentrations and 2H enrichments in microvolumes of neonatal blood using gas chromatography–mass spectrometry , 2008, Analytical and bioanalytical chemistry.

[13]  A. Palamara,et al.  Antiviral and immunomodulatory properties of new pro-glutathione (GSH) molecules. , 2006, Current medicinal chemistry.

[14]  D. Giustarini,et al.  Biomarkers of oxidative damage in human disease. , 2006, Clinical chemistry.

[15]  D. Townsend,et al.  The importance of glutathione in human disease. , 2003, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[16]  N. Holbrook,et al.  Oxidants, oxidative stress and the biology of ageing , 2000, Nature.

[17]  P. Fürst,et al.  Glutathione and glutathione metabolites in small tissue samples and mucosal biopsies. , 2000, Clinical chemistry.

[18]  Shelly C. Lu,et al.  Regulation of Hepatic Glutathione Synthesis , 1998, Seminars in liver disease.