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Validation of a simplified procedure for convenient and rapid quantification of reduced and oxidized glutathione in human plasma by liquid chromatography tandem mass spectrometry analysis
Author(s) -
Enomoto Addison C.,
Schneider Erik,
McKin Toni,
Goldfine Howard,
Levy Mark A.
Publication year - 2020
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.4854
Subject(s) - chemistry , glutathione , chromatography , glutathione disulfide , analyte , quantitative analysis (chemistry) , tandem mass spectrometry , liquid chromatography–mass spectrometry , detection limit , matrix (chemical analysis) , high performance liquid chromatography , sample preparation , mass spectrometry , biochemistry , enzyme
Endogenous glutathione (GSH) and glutathione disulfide (GSSG) status is highly sensitive to oxidative conditions and have broad application as a surrogate indicator of redox status in vivo. Established methods for GSH and GSSG quantification in whole blood display limited utility in human plasma, where GSH and GSSG levels are ~3–4 orders of magnitude below those observed in whole blood. This study presents simplified sample processing and analytical LC–MS/MS approaches exhibiting the sensitivity and accuracy required to measure GSH and GSSG concentrations in human plasma samples, which after 5‐fold dilution to suppress matrix interferences range from 200 to 500 n m (GSH) and 5–30 n m (GSSG). The utility of the methods reported herein is demonstrated by assay performance and validation parameters which indicate good sensitivity [lower limits of quantitation of 4.99 n m (GSH) and 3.65 n m (GSSG), and high assay precision (intra‐assay CVs 3.6 and 1.9%, and inter‐assay CVs of 7.0 and 2.8% for GSH and GSSG, respectively). These methods also exhibited exceptional recovery of analyte‐spiked plasma samples (98.0 ± 7.64% for GSH and 98.5 ± 12.7% for GSSG). Good sample stability at −80°C was evident for GSH for up to 55 weeks and GSSG for up to 46 weeks, with average CVs <15 and <10%, respectively.