Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume 37, Number
12, December 1999, pp. 469-476
Measurement
of Thiols in Human Plasma Using Liquid Chromatography with Precolumn Derivatization
and Fluorescence Detection
Jean-Frédéric
Salazar, Heike Schorr, Wolfgang Herrmann, Bernard Herbeth, Gérard Siest,
and Pierre LeroyA
liquid chromatography (LC) method for the simultaneous measurement of the main
low molecular mass thiols (i.e., cysteine, cysteinylglycine, homocysteine, and
glutathione) in human plasma is described. The sample treatment consists of the
reduction of disulfide bounds with tri-n-butylphosphine and protein precipitation
with trichloroacetic acid followed by precolumn derivatization with a thiol-selective
fluorogenic reagent (7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide). The structure
of thiol derivatives is assessed using electrospray ionization–mass spectrometry
(MS). The stability of resulting adducts in acidic medium (24 h at 10°C) allows
the automation of the technique and a high throughput of samples (approximately
50 per day). Separation is complete within 12 min using isocratic reversed-phase
mode, and detection is operated by spectrofluorimetry (lex = 385 nm and lem =
515 nm). Quantitation is performed by an internal standardization mode using thioglycolic
acid. The LC method is fully validated, and homocysteine concentrations obtained
in plasma samples are compared with values measured using either fluorescence
polarization immunoassay or capillary gas chromatography–MS; a good correlation
is observed between LC and both methods. The method has been applied in daily
use to a large-scale study in a human healthy population, and some resulting data
are discussed.
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