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Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography

Nuhu, Faisal; Gordon, Andrew; Sturmey, Roger; Seymour, Anne Marie; Bhandari, Sunil


Faisal Nuhu

Andrew Gordon

Anne Marie Seymour

Sunil Bhandari


Background: Maintenance of the ratio of glutathione in the reduced (GSH) and oxidised (GSSG) state in cells is important in redox control, signal transduction and gene regulation, factors that are altered in many diseases. The accurate and reliable determination of GSH and GSSG simultaneously is a useful tool for oxidative stress determination. Measurement is limited primarily to the underestimation of GSH and overestimation GSSG as a result of auto-oxidation of GSH. The aim of this study was to overcome this limitation and develop, optimise and validate a reverse-phase high performance liquid chromatographic (HPLC) assay of GSH and GSSG for the determination of oxidant status in cardiac and chronic kidney diseases. Methods: Fluorescence detection of the derivative, glutathione-O-pthaldialdehyde (OPA) adduct was used. The assay was validated by measuring the stability of glutathione and glutathione-OPA adduct under conditions that could affect the reproducibility including reaction time and temperature. Linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), recovery and extraction efficiency and selectivity of the method were assessed. Results: There was excellent linearity for GSH (r2 = 0.998) and GSSG (r2 = 0.996) over concentration ranges of 0.1 μM–4 mM and 0.2 μM–0.4 mM respectively. The extraction of GSH from tissues was consistent and precise. The limit of detection for GSH and GSSG were 0.34 μM and 0.26 μM respectively whilst their limits of quantification were 1.14 μM and 0.88 μM respectively. Conclusion: These data validate a method for the simultaneous measurement of GSH and GSSG in samples extracted from biological tissues and offer a simple determination of redox status in clinical samples.


Nuhu, F., Gordon, A., Sturmey, R., Seymour, A. M., & Bhandari, S. (2020). Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography. Molecules, 25(18), Article 4196.

Journal Article Type Article
Acceptance Date Sep 8, 2020
Online Publication Date Sep 13, 2020
Publication Date Sep 2, 2020
Deposit Date Sep 14, 2020
Publicly Available Date Sep 15, 2020
Journal Molecules
Electronic ISSN 1420-3049
Publisher MDPI
Peer Reviewed Peer Reviewed
Volume 25
Issue 18
Article Number 4196
Keywords Glutathione analysis; Oxidative stress; HPLC; GSH; GSSG
Public URL
Publisher URL


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Copyright Statement
©2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (

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