Clinical Chemistry
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Clinical Chemistry 51: 1007-1013, 2005. First published April 21, 2005; 10.1373/clinchem.2004.045443
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Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 2005;51:1007-1013.)
© 2005 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Speciation and Quantification of Thiols by Reversed-Phase Chromatography Coupled with On-Line Chemical Vapor Generation and Atomic Fluorescence Spectrometric Detection: Method Validation and Preliminary Application for Glutathione Measurements in Human Whole Blood

Emilia Bramanti1,a, Cecilia Vecoli2, Danilo Neglia2, Maria Paola Pellegrini2, Giorgio Raspi1 and Renata Barsacchi2,3

1 Italian National Research Council, Istituto per i Processi Chimico-Fisici, Laboratory of Instrumental Analytical Chemistry, Pisa, Italy.
2 Italian National Research Council, Istituto di Fisiologia Clinica, Pisa, Italy.
3 Department of Physiology and Biochemistry, University of Pisa, Pisa, Italy.

aAddress correspondence to this author at: Italian National Research Council, Istituto per i Processi Chimico-Fisici, Laboratory of Instrumental Analytical Chemistry, Via G. Moruzzi 1, 56124 Pisa, Italy. Fax 39-050-315-2555; e-mail emilia{at}ipcf.cnr.it.

Background: We developed a sensitive, specific method for the low–molecular-mass thiols cysteine, cysteinylglycine, glutathione, and homocysteine and validated the method for measurement of glutathione in blood.

Methods: The technique was based on reversed-phase chromatography (RPC) coupled on line with cold vapor generation atomic fluorescence spectrometry (CVGAFS). Thiols were derivatized before introduction on the column by use of a p-hydroxymercuribenzoate (PHMB) mercurial probe and separated as thiol-PHMB complexes on a Vydac C4 column. Postcolumn on-line reaction of derivatized thiols with bromine allowed rapid conversion of the thiol-PHMB complexes to inorganic mercury with recovery of 100 (2)% of the sample. HgII was selectively detected by atomic fluorescence spectrometry in an Ar/H2 miniaturized flame after sodium borohydride reduction to Hg0.

Results: The relationship between thiol-PHMB complex concentration and peak area (CVGAFS signal) was linear over the concentration range 0.01–1400 µmol/L (injected). The detection limits were 1, 1, 0.6, and 0.8 nmol/L for cysteine, cysteinylglycine, homocysteine, and glutathione in the injected sample, respectively. The CVs for thiols were 1.5%–2.2% for calibrator solutions and 2.1% and 3.0% for real samples. The RPC-CVGAFS method allowed speciation of glutathione (reduced and oxidized) in human whole blood from healthy donors and from the coronary sinus of patients with idiopathic dilated cardiomyopathy during and after chronotropic stress.

Conclusion: The RPC-CVGAFS method could be used to measure reduced and oxidized glutathione in human whole blood as disease biomarkers.






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