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 journalArticle

71 Citations (Scopus)

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

Fingerprint

Glutathione Disulfide
Liquid chromatography
Mass spectrometry
Glutathione
Blood
Assays
Ethylmaleimide
Acetic Acid
Recovery
Oxidation

Keywords

  • Glutathione

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

@article{28d08946892c465ea6a2ee9b7fa84a81,
title = "Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry",
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.",
keywords = "Glutathione",
author = "Emanuela Camera and Mariarosaria Rinaldi and Stefania Briganti and Mauro Picardo and Salvatore Fanali",
year = "2001",
month = "6",
day = "5",
doi = "10.1016/S0378-4347(01)00081-0",
language = "English",
volume = "757",
pages = "69--78",
journal = "Journal of Chromatography B: Biomedical Sciences and Applications",
issn = "1387-2273",
publisher = "Elsevier BV",
number = "1",

}

TY - JOUR

T1 - Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry

AU - Camera, Emanuela

AU - Rinaldi, Mariarosaria

AU - Briganti, Stefania

AU - Picardo, Mauro

AU - Fanali, Salvatore

PY - 2001/6/5

Y1 - 2001/6/5

N2 - 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.

AB - 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.

KW - Glutathione

UR - http://www.scopus.com/inward/record.url?scp=0035811248&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035811248&partnerID=8YFLogxK

U2 - 10.1016/S0378-4347(01)00081-0

DO - 10.1016/S0378-4347(01)00081-0

M3 - Article

C2 - 11419750

AN - SCOPUS:0035811248

VL - 757

SP - 69

EP - 78

JO - Journal of Chromatography B: Biomedical Sciences and Applications

JF - Journal of Chromatography B: Biomedical Sciences and Applications

SN - 1387-2273

IS - 1

ER -