“One-pot” ethyl chloroformate derivatization and liquid-liquid extraction of reduced glutathione in erythrocyte and its quantitative GC–MS analysis

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1070; pp. 15 - 22
• Main Authors: Manca, Alessandra, Alladio, Eugenio, Massarenti, Paola, Puccinelli, M. Paola, De Francesco, Antonella, Del Grosso, Erika, Mengozzi, Giulio, Pazzi, Marco, Vincenti, Marco
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2017
• Subjects: Chloroformate derivatization; clinical trials; derivatization; Erythrocytes; Erythrocytes - chemistry; ethanol; Formic Acid Esters - chemistry; Gas chromatography-mass spectrometry; Gas Chromatography-Mass Spectrometry - methods; Glutathione; Glutathione - blood; Glutathione - chemistry; Glutathione - isolation & purification; Humans; kwashiorkor; Limit of Detection; Linear Models; Liquid-Liquid Extraction - methods; liquids; metabolites; patients; Pilot Projects; pyridines; Reproducibility of Results; Chloroformate derivatization; Gas chromatography-mass spectrometry; Erythrocytes; Glutathione
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2017.10.030

•Development of “one-pot” ECF-EtOH derivatization and LLE sample preparation method.•For GC/MS analysis of reduced glutathione (GSH) and hGSH in erythrocytes.•Comprehensive validation of the novel analytical methodology.•Feasibility for clinical investigation of the oxidative status: case studies. A simple “one-pot” derivatization and liquid–liquid extraction (LLE) procedure was developed for GC–MS analysis of reduced glutathione (GSH) analysis in erythrocytes. The metabolite was extracted by 5% (w/v) TCA, the supernatant treated with ECF and ethanol-pyridine media, the derivative separated and detected by gas chromatography-mass spectrometry using a short non-polar capillary GC column at a high column-head pressure. Total analysis time was 11min. The process was optimized by a Design of Experiment. The method was validated showing a good linearity over the 25.4–813.4μM concentration range, providing satisfactory results in terms of intra-day and inter-day precision as well as an optimal accuracy. The new method was evaluated in a pilot study involving patients with severe protein malnutrition. Comparison of this group with a group of healthy subjects revealed significantly lower GSH concentrations in erythrocytes in the former, thus proving that the described GC–MS method could be employed for fast and simple GSH analysis in clinical studies.