Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry

Emanuela Camera, Mariarosaria Rinaldi, Stefania Briganti, Mauro Picardo, Salvatore Fanali

Research output: Contribution to journalArticlepeer-review

Abstract

We developed a sensitive and specific liquid chromatography-electrospray mass spectrometric (HPLC-ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile-1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 μM for GSH and GSSG, respectively). Good linearity was reached in the 0.01-20 μM range for GSH and 0.05-20 μM for GSSG. The mean recoveries of GSH and GSSG were 98.5-100.6% and 105.8-111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.

Original languageEnglish
Pages (from-to)69-78
Number of pages10
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume757
Issue number1
DOIs
Publication statusPublished - Jun 5 2001

Keywords

  • Glutathione

ASJC Scopus subject areas

  • Chemistry(all)

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