A rapid and simple method, using pre-column fluorescent labeling, followed by high pressure liquid chromatography (HPLC), for the determination of total concentration of thiols (homocysteine, cysteine, cystinylglycine, and glutathione) in small-volume, pediatric samples, is presented.
Thiols in plasma were first reduced with tri-n-butylphosphine, and the resulting free-SH groups were labeled with the fluorescent compound 4- (aminosulfonyl) - 7-fluoro-2,1,3-benzoxadiazole (ABD-F).
The ABD-thiols were desorbed from plasma proteins by precipitating proteins with TCA, and then fractionated by reverse-phase chromatography.
Peak areas were used to determine concentrations in the unknowns.
Baseline resolution of all four thiols was obtained in about 12 minutes.
This method requires a maximum of 75 muL or less of the sample.
It is specific to thiols, and chromatograms do not show any other peaks.
It is sensitive to about 500 pmol/L, and is linear to at least 300 mumol/L of each of the thiols.
Within-and between-day CV's were less than 4%. Reference ranges for children up to 18 years of age (mean ± SD), were : 8.9<2.9, and 232 ± 56 umol/L for total homocysteine, and total cysteine, respectively.
No sex-or age-dependent changes were observed in children up to 18 years of age of reference values for total homocysteine or total cysteine.
Mots-clés Pascal : Analyse chimique, Analyse quantitative, Surveillance biologique, Nourrisson, Homme, Enfant, Adolescent, Liquide biologique, Sang, Plasma sanguin, Aminoacide soufré, Homocystéine, Thiol, Cystéine, Glutathion, Peptide soufre, Dipeptide, Dérivatisation, Précolonne, Préparation échantillon, Chromatographie HPLC, Détecteur fluorescence, Glycine(cystyl), 2,1,3-Benzooxadiazole-4-sulfonique acide(7-fluoro) amide
Mots-clés Pascal anglais : Chemical analysis, Quantitative analysis, Biological monitoring, Infant, Human, Child, Adolescent, Biological fluid, Blood, Blood plasma, Sulfur containing aminoacid, Thiol, Cysteine, Glutathione, Sulfur peptide, Dipeptides, Derivatization, Precolumn, Sample preparation, HPLC chromatography, Fluorescence detector
Notice produite par :
Inist-CNRS - Institut de l'Information Scientifique et Technique
Cote : 97-0287683
Code Inist : 002B30A01A2. Création : 15/07/1997.