Validation and optimisation of reduced glutathione quantification in erythrocytes by means of a coulometric high-performance liquid chromatography analytical method.

Abstract

Glutathione (GSH), a tripeptide that consists of cysteine, glutamate and glycine, is present in all mammalian tissues in the millimolar range. Besides having numerous cellular functions, GSH is an important antioxidant and is considered a valuable biomarker in evaluating oxidative stress. This paper provides a sensitive analytical method using HPLC-ECD to quantify GSH in erythrocytes, validated using the ICH guidelines for Bioanalytical Method Validation. The sample preparation was optimised using centrifugal filtration and a hypotonic phosphate buffer for extracting GSH from erythrocytes. HPLC-ECD parameters were adjusted to allow a fast, reversed phase, isocratic separation in 10 min. The detector response was linear between 0.3 and 9.5 μg/mL with a satisfactory regression coefficient and a LOQ of 0.11 μg/mL. Intra- and inter-day repeatability ranged between 1.10% and 8.57% with recoveries ranging from 94.3% to 106.0%. Dilution integrity, benchtop, freeze-thaw and long-term stability were investigated. Samples were stable for up to 6 months at -80°C. This method has a good linear response and is repeatable, precise and accurate. It minimises GSH auto-oxidation using a centrifugal filter during sample preparation, instead of acidification. Therefore, this analytical method is suitable for quantifying GSH in erythrocytes as a marker of oxidative stress.