Glutathione (GSH) is an important non-protein thiol compound that plays a key role in maintaining cellular homeostasis as well as protecting cells against oxidative stress. Glutathione depletion has been identified in various disease processes such as cardiovascular diseases, carcinogenesis and aging. Under pathological conditions GSH is converted into glutathione disulfide (GSSG) the oxidized form, resulting in a decreased GSH/GSSG ratio. As this ratio is considered to be an early indicator of oxidative stress and/or disease risk, it is important to measure both GSH and GSSG in whole blood samples which may reflect glutathione status in other less accessible tissues. An analytical method based on liquid-chromatography with positive electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC-MS/MS) was developed for the determination of both GSH and GSSG in human whole blood. Separation of analytes was conducted on a Luna PFP(2) analytical column (100x2.0 mm, 3 µm; Phenomenex, USA). A Thermo TSQ Quantum Access triple quadrupole mass spectrometer coupled with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 308.1→76.2, 84.2, 161.9 (GSH) and 613.2→230.5, 234.6, 354.8 (GSSG) used for quantification. The assay was fully validated and found to perform well in terms of validation parameters. The method is linear over a concentration range of 0.01 to 50 µM with a lower limit of quantitation (LLOQ) of 0.01 µM for both compounds. The intra-day precision values for all quality control (QC) samples were 2.7% for GSH and 5.4% for GSSG. The inter-day assay precision values for all QC samples were 6.7% for GSH and 6.5% for GSSG. The method also showed adequate accuracy values for both analytes. The new method was compared with the HPLC coupled with electrochemical detector (HPLC-ECD) assay which we routinely use, and the two assays were found to show a good agreement. In conclusion although both methods are reliable, easy and fast to perform, the major benefits of the LC-MS/MS are related to the small whole blood volume required (only one-twentieth if that needed by HPLC-ECD assay) and to the higher sensitivity and selectivity, with a better precision and accuracy, thus allowing the quantification of both analytes also in low-concentration samples.

Direct measurement of reduced and oxidized glutathione in human blood by liquid chromatography tandem mass spectrometry – comparison with HPLC with electrochemical detection method / I. Squellerio, D. Caruso, F. Veglia, G. Falcucci, E. Tremoli, V.M. Cavalca. ((Intervento presentato al 6. convegno Massa 2010 - Pharmaday tenutosi a Milano nel 2010.

Direct measurement of reduced and oxidized glutathione in human blood by liquid chromatography tandem mass spectrometry – comparison with HPLC with electrochemical detection method

D. Caruso
Secondo
;
E. Tremoli
Penultimo
;
V.M. Cavalca
Ultimo
2010

Abstract

Glutathione (GSH) is an important non-protein thiol compound that plays a key role in maintaining cellular homeostasis as well as protecting cells against oxidative stress. Glutathione depletion has been identified in various disease processes such as cardiovascular diseases, carcinogenesis and aging. Under pathological conditions GSH is converted into glutathione disulfide (GSSG) the oxidized form, resulting in a decreased GSH/GSSG ratio. As this ratio is considered to be an early indicator of oxidative stress and/or disease risk, it is important to measure both GSH and GSSG in whole blood samples which may reflect glutathione status in other less accessible tissues. An analytical method based on liquid-chromatography with positive electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC-MS/MS) was developed for the determination of both GSH and GSSG in human whole blood. Separation of analytes was conducted on a Luna PFP(2) analytical column (100x2.0 mm, 3 µm; Phenomenex, USA). A Thermo TSQ Quantum Access triple quadrupole mass spectrometer coupled with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 308.1→76.2, 84.2, 161.9 (GSH) and 613.2→230.5, 234.6, 354.8 (GSSG) used for quantification. The assay was fully validated and found to perform well in terms of validation parameters. The method is linear over a concentration range of 0.01 to 50 µM with a lower limit of quantitation (LLOQ) of 0.01 µM for both compounds. The intra-day precision values for all quality control (QC) samples were 2.7% for GSH and 5.4% for GSSG. The inter-day assay precision values for all QC samples were 6.7% for GSH and 6.5% for GSSG. The method also showed adequate accuracy values for both analytes. The new method was compared with the HPLC coupled with electrochemical detector (HPLC-ECD) assay which we routinely use, and the two assays were found to show a good agreement. In conclusion although both methods are reliable, easy and fast to perform, the major benefits of the LC-MS/MS are related to the small whole blood volume required (only one-twentieth if that needed by HPLC-ECD assay) and to the higher sensitivity and selectivity, with a better precision and accuracy, thus allowing the quantification of both analytes also in low-concentration samples.
2010
Settore BIO/10 - Biochimica
Settore BIO/14 - Farmacologia
Settore BIO/12 - Biochimica Clinica e Biologia Molecolare Clinica
Direct measurement of reduced and oxidized glutathione in human blood by liquid chromatography tandem mass spectrometry – comparison with HPLC with electrochemical detection method / I. Squellerio, D. Caruso, F. Veglia, G. Falcucci, E. Tremoli, V.M. Cavalca. ((Intervento presentato al 6. convegno Massa 2010 - Pharmaday tenutosi a Milano nel 2010.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/163008
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