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  • Title: [Determination of total plasma homocysteine and other aminothiols by liquid chromatography coupled to the detection by fluorescence].
    Author: Jacob N, Guillaume L, Garçon L, Foglietti MJ.
    Journal: Ann Biol Clin (Paris); 1997; 55(6):583-91. PubMed ID: 9499919.
    Abstract:
    Plasma concentrations of homocysteine, cysteine, cysteinylglycine and glutathione were measured using a HPLC technique with fluorescence detection of the derivatives obtained with 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide (ABD-F). Blood was drawn into chilled EDTA-evacuated tubes. After centrifugation at 4 degrees C without delay, plasma samples were kept frozen at -20 degrees C until analysis. Reduction of protein bound aminothiols and disulfides standards was achieved with tri-n-butylphosphine. N-acetylcysteine was used as internal standard. After protein precipitation, derivatization was carried out at pH 8.0 and 50 degrees C for 20 min. Stability of ABD-thiols was ensured for at least 5 days by lowering pH to 2. Derivatives were separated by isocratic elution on a Waters mu Bondapak C18 column (10 microns, 3.9 x 300 mm) with 0.1 M phosphate buffer pH 3.2 containing 10% acetonitrile. Excitation and emission wavelengths were 385 and 515 nm. Retention times were 4.9, 5.8, 7.3, 9.9 and 20.1 min respectively for cysteine, cysteinylglycine, homocysteine, glutathione and N-acetylcysteine. Peaks were quantified by comparison to a standard curve prepared by plotting peak height versus the different levels of known standard solutions after normalization with internal standard. Between-run CVs varied from 5 to 8.5%. The detection limit was < 0.5 mumol/l for homocysteine and glutathione. In plasma samples from healthy subjects, concentration of homocysteine was higher in men than in women (11.0 +/- 2.9 versus 9.2 +/- 2.7 mumol/l, p < 0.01). These values are similar to those obtained with other widely used methods.
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