A stable-isotope HPLC–MS/MS method to simplify storage of human whole blood samples for glutathione assay

BackgroundGlutathione is the principal non-protein tripeptide thiol present in most mammalian cells and plays an important role in the redox status of biological systems. The accurate assessment of reduced glutathione (GSH) status as a reliable index of oxidative stress is of research and clinical significance. GSH undergoes rapid oxidation after sample collection and this presents a challenge.MethodsValidation of an HPLC–MS/MS assay is reported. Storage stability using four variants of a methanolic precipitation with addition of stable isotope internal standard at collection is compared to l-serine borate/EDTA with perchloric acid precipitation (SBPE).ResultsPrecipitation with methanol and addition of stable isotope on sample collection, combined with storage in solution at −70 °C showed superior storage stability to SBPE and other variants of the methanolic precipitation method up to 99 days.ConclusionsThe combination of stable isotope with methanolic precipitation at collection, with assay by HPLC–MS/MS provides superior results after storage of whole blood samples for at least 99 days.

► A n HPLC–MS/MS assay for determination of reduced glutathione is presented.
► Deuterated internal standard with precipitation at sample collection allows storage for 99 days.
► Improved storage compared to EDTA/perchloric acid/serine borate pre-treatment.