Direct glutathione quantification in human blood by LC-MS/MS: Comparison with HPLC with electrochemical detection

Isabella Squellerio, Donatella Caruso, Benedetta Porro, Fabrizio Veglia, Elena Tremoli, Viviana Cavalca

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Glutathione plays a central role in the defence against oxidative damage and in signaling pathways. Upon oxidation the reduced glutathione (GSH) is transformed to glutathione disulfide (GSSG). The concentration of GSH and GSSG in whole blood samples and their ratios is useful indicator of the oxidative stress status and/or disease risk. Here we describe a liquid-chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) and we present the results of its comparison with a high-performance liquid-chromatographic method with electrochemical detection (HPLC-ECD). The method performed well in terms of validation parameters, i.e. linear range (0.01-50μM for both GSH and GSSG), precision (intra- and inter-day coefficients of variation were less than 10% for both GSH and GSSG), accuracy (bias% varied between -2.1 and 7.9% for both analytes), quantification limits (LLOQs were 0.5μM and 0.0625μM for GSH and GSSG respectively). Furthermore the LC-MS/MS method showed a good agreement with the HPLC-ECD assay. However, major benefits of LC-MS/MS are the improved selectivity, precision and accuracy, the higher sensitivity and the unaltered capacity of detection with time in contrast to ECD.

Original languageEnglish
Pages (from-to)111-118
Number of pages8
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume71
DOIs
Publication statusPublished - Dec 2012

Fingerprint

Glutathione Disulfide
Glutathione
Blood
High Pressure Liquid Chromatography
Liquids
Oxidative stress
Tandem Mass Spectrometry
Mass spectrometry
Assays
Oxidative Stress
Oxidation

Keywords

  • Glutathione
  • High-performance liquid-chromatographic with electrochemical detector
  • Liquid-chromatographic tandem mass spectrometry

ASJC Scopus subject areas

  • Analytical Chemistry
  • Drug Discovery
  • Pharmaceutical Science
  • Spectroscopy
  • Clinical Biochemistry

Cite this

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title = "Direct glutathione quantification in human blood by LC-MS/MS: Comparison with HPLC with electrochemical detection",
abstract = "Glutathione plays a central role in the defence against oxidative damage and in signaling pathways. Upon oxidation the reduced glutathione (GSH) is transformed to glutathione disulfide (GSSG). The concentration of GSH and GSSG in whole blood samples and their ratios is useful indicator of the oxidative stress status and/or disease risk. Here we describe a liquid-chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) and we present the results of its comparison with a high-performance liquid-chromatographic method with electrochemical detection (HPLC-ECD). The method performed well in terms of validation parameters, i.e. linear range (0.01-50μM for both GSH and GSSG), precision (intra- and inter-day coefficients of variation were less than 10{\%} for both GSH and GSSG), accuracy (bias{\%} varied between -2.1 and 7.9{\%} for both analytes), quantification limits (LLOQs were 0.5μM and 0.0625μM for GSH and GSSG respectively). Furthermore the LC-MS/MS method showed a good agreement with the HPLC-ECD assay. However, major benefits of LC-MS/MS are the improved selectivity, precision and accuracy, the higher sensitivity and the unaltered capacity of detection with time in contrast to ECD.",
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author = "Isabella Squellerio and Donatella Caruso and Benedetta Porro and Fabrizio Veglia and Elena Tremoli and Viviana Cavalca",
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T1 - Direct glutathione quantification in human blood by LC-MS/MS

T2 - Comparison with HPLC with electrochemical detection

AU - Squellerio, Isabella

AU - Caruso, Donatella

AU - Porro, Benedetta

AU - Veglia, Fabrizio

AU - Tremoli, Elena

AU - Cavalca, Viviana

PY - 2012/12

Y1 - 2012/12

N2 - Glutathione plays a central role in the defence against oxidative damage and in signaling pathways. Upon oxidation the reduced glutathione (GSH) is transformed to glutathione disulfide (GSSG). The concentration of GSH and GSSG in whole blood samples and their ratios is useful indicator of the oxidative stress status and/or disease risk. Here we describe a liquid-chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) and we present the results of its comparison with a high-performance liquid-chromatographic method with electrochemical detection (HPLC-ECD). The method performed well in terms of validation parameters, i.e. linear range (0.01-50μM for both GSH and GSSG), precision (intra- and inter-day coefficients of variation were less than 10% for both GSH and GSSG), accuracy (bias% varied between -2.1 and 7.9% for both analytes), quantification limits (LLOQs were 0.5μM and 0.0625μM for GSH and GSSG respectively). Furthermore the LC-MS/MS method showed a good agreement with the HPLC-ECD assay. However, major benefits of LC-MS/MS are the improved selectivity, precision and accuracy, the higher sensitivity and the unaltered capacity of detection with time in contrast to ECD.

AB - Glutathione plays a central role in the defence against oxidative damage and in signaling pathways. Upon oxidation the reduced glutathione (GSH) is transformed to glutathione disulfide (GSSG). The concentration of GSH and GSSG in whole blood samples and their ratios is useful indicator of the oxidative stress status and/or disease risk. Here we describe a liquid-chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) and we present the results of its comparison with a high-performance liquid-chromatographic method with electrochemical detection (HPLC-ECD). The method performed well in terms of validation parameters, i.e. linear range (0.01-50μM for both GSH and GSSG), precision (intra- and inter-day coefficients of variation were less than 10% for both GSH and GSSG), accuracy (bias% varied between -2.1 and 7.9% for both analytes), quantification limits (LLOQs were 0.5μM and 0.0625μM for GSH and GSSG respectively). Furthermore the LC-MS/MS method showed a good agreement with the HPLC-ECD assay. However, major benefits of LC-MS/MS are the improved selectivity, precision and accuracy, the higher sensitivity and the unaltered capacity of detection with time in contrast to ECD.

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