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

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.

► An LC–MS/MS assay for the determination of GSH and GSSG in whole blood is described.
► The method was compared with an HPLC assay with electrochemical detection (HPLC-ECD).
► LC–MS/MS had higher selectivity and lower variability in comparison with HPLC-ECD.
► The new method allows the quantification of both analytes also in low-concentration samples.