Direct glutathione quantification in human blood by LC–MS/MS: comparison with HPLC with electrochemical detection

Abstract

Glutathione plays a central role in the defence against oxidative damage and in signaling pathways. Upon oxidation the reduced glutathione (GSH) is transformed to glutathione disulfide (GSSG). The concentration of GSH and GSSG in whole blood samples and their ratios is useful indicator of the oxidative stress status and/or disease risk. Here we describe a liquid-chromatographic method coupled with tandem mass spectrometry (LC–MS/MS) and we present the results of its comparison with a high-performance liquid-chromatographic method with electrochemical detection (HPLC-ECD). The method performed well in terms of validation parameters, i.e. linear range (0.01–50 μM for both GSH and GSSG), precision (intra- and inter-day coefficients of variation were less than 10% for both GSH and GSSG), accuracy (bias% varied between −2.1 and 7.9% for both analytes), quantification limits (LLOQs were 0.5 μM and 0.0625 μM for GSH and GSSG respectively). Furthermore the LC–MS/MS method showed a good agreement with the HPLC-ECD assay. However, major benefits of LC–MS/MS are the improved selectivity, precision and accuracy, the higher sensitivity and the unaltered capacity of detection with time in contrast to ECD.

Introduction

Glutathione (GSH, γ-l-glutamyl-l-cysteinyl-glycine) is an important non-protein thiol compound and plays a key role in maintaining cellular homeostasis as well as protecting cells against oxidative stress. Glutathione depletion has been identified in various disease processes such as cardiovascular diseases, carcinogenesis and aging [1], [2]. Under pathological conditions GSH is converted into glutathione disulfide (GSSG) the oxidized form, resulting in a decreased GSH/GSSG ratio. As this ratio is considered to be an early indicator of oxidative stress and/or disease risk [3], it is important to measure both GSH and GSSG in whole blood samples which may reflect glutathione status in other less accessible tissues [4]. As previously demonstrated [4], [5] the measurement of glutathione in biological samples requires caution in order to prevent assay artifacts or data misinterpretation. The major weak points in the glutathione determination are GSH autoxidation ease, the enzymatic proteolysis of GSH by γ-glutamyl transferase (γGT) and the rapid GSSG degradation by proteolysis [6]. GSH, rapidly oxidizes non-enzymatically at pH values greater than 7, and γGT has an optimum of activity at neutral pH. Thus, it is suggested to maintain the pH of media in which GSH is processed below pH 7 [7].

Over the years, a wide variety of analytical methods have been introduced for the determination of glutathione in biological samples based on different detection and separation techniques [8]. Early techniques including enzymatic [9], [10], fluorimetric [11], colorimetric [12] and bioluminescence based [13] assays had poor specificity, inadequate detection limits or low reproducibility. More recently, several procedures using high-performance liquid chromatography have been developed. These methods were developed in the attempt to get over these limits by using either UV absorbance evaluation [14], [15], [16] or fluorescence measurement after derivatization [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], but most of these require pre- or postcolumn derivatization and present complicated and time-consuming protocols or insufficient sensitivity for small samples [17]. More importantly several methods are useful only in detecting thiols such as GSH and a second assay is required for the determination of GSSG.

Progress has been made by HPLC coupled with electrochemical detector (HPLC-ECD) which avoids the need for derivatization and allows the simultaneous determination of both compounds with relatively short analysis times [27], [28], [29], [30], [31]. However, a major limitation of this approach includes the electrochemical detection loss of signal after repeated analysis. Carro-Ciampi et al. [32] and Stempak et al. [33] noted a 50% loss in the GSSG signal after only 100 injections.

Recent developments in mass spectrometric (MS) technology, coupled with liquid chromatography (LC) have provided new tools for more sensitive and selective glutathione quantitation [34]. In LC–MS methods, the autoxidation of GSH has been prevented by blocking the thiol group with iodoacetic acid [35], [36], Ellman/s reagent [37], ABD-F [38] or N-ethylmaleimide [39], [40], [41], [42], [43]. So far, only few studies have attempted to simultaneously measure GSH and GSSG without adding any derivatization reagent [44], [45], [46], [47]. In the current work we present a simple and direct LC tandem mass spectrometry (LC–MS/MS) method to accurately measure the whole blood GSH and GSSG contents without any derivatization step. Furthermore we compared the new method with the HPLC-ECD assay which we routinely use.