HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue

R. Paroni, E. De Vecchi, G. Cighetti, C. Arcelloni, I. Fermo, A. Grossi, P. Bonini

Research output: Contribution to journalArticlepeer-review

Abstract

This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathione in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined after reduction with dithiothreitol and protein precipitation with perchloric acid (PCA). A preliminary selective blockage of free sulfhydryl groups with N- ethylmaleimide was necessary to evaluate the different oxidized forms. The assay showed high sensitivity (0.999). Samples, after PCA acidification, were stable at room temperature and 4°C for 3 days, and at -20°C and -80°C for >1 month. The method (involving automated derivatization) not only is very rapid and simple but also allows immediate processing of many different biological samples.

Original languageEnglish
Pages (from-to)448-454
Number of pages7
JournalClinical Chemistry
Volume41
Issue number3
Publication statusPublished - 1995

Keywords

  • chromatography, reversed-phase
  • fluorometry
  • sample handling

ASJC Scopus subject areas

  • Clinical Biochemistry

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