A new LC–MS/MS method for the clinical determination of reduced and oxidized glutathione from whole blood

Reduced levels of glutathione (γ-glutamylcysteinylglycine, GSH) and the ratio of GSH to glutathione disulfide (GSSG) can serve as important indicators of oxidative stress and disease risk. Measured concentrations of GSH and GSSG vary widely between laboratories, largely due to the instability of GSH during sample handling and variables arising from different analytical methods. We have developed a simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for measuring whole blood GSH and GSSG that minimizes preanalytic and analytic variability, reliably eliminates interference from ion suppression, and can easily be implemented in clinical laboratories. Samples were deproteinized with sulfosalicylic acid (SSA) and derivatized with N-ethylmaleimide (NEM) in a single preparative step, and the resulting supernatants combined with stable-isotope internal standards (GSH-13C, 15N-NEM and GSSG-13C,15N), subjected to chromatographic separation using a Hypercarb column, and analyzed by MS/MS in the positive-ion mode. Results showed excellent linearity for both GSH and GSSG over the ranges of physiologic normal, with inter- and intra-assay CVʹs of 3.1–4.3% and accuracy between 95% and 101%. The lower limits of detection (LLOD) were 0.4 μM for GSH and 0.1 μM for GSSG and the lower limits of quantitation (LLOQ) were 1.5 μM for GSH and 0.1 μM for GSSG. Derivatized samples are stable for at least 3 years when stored at −80 °C, and underivatized samples for at least 24 h at either 4 °C or room temperature. Reference intervals were determined for 59 control samples, and were (mean ± SD): GSH 900 ± 140 μM; GSSG 1.17 ± 0.43 μM; GSH/GSSG 880 ± 370.