Speciation and quantitative determination of thiols by Reversed Phase Coupled with On Line Chemical Vapour Generation and Atomic Fluorescence Spectrometric Detection (RP-CVGAFS). Method validation and preliminary applications

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 mol/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 RPCCVGAFS
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.