Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

The determination of low-molecular-mass thiols with 4-(hydroxymercuric)benzoic acid as a tag using HPLC coupled online with UV/HCOOH-induced cold vapor generation AFS.

PMID 19524490


An alternative analytical method was established for simultaneous determination of main urinary low-molecular-mass (LMM) thiols including cysteine (Cys), cysteinylglycine (Cys-Gly), homocysteine (HCys), gamma-glutamyl cysteine (gamma-Glu-Cys) and glutathione (GSH) as well as N-acetylcysteine (NAC) using RPLC coupled on line with UV/HCOOH-induced cold vapor generation atomic fluorescence spectrometry (UV/HCOOH-CVG-AFS) with 4-(hydroxymercuric)benzoic acid (PHMB) as a tag. The LMM thiols were stabilized and labeled by PHMB allowing the determination of reduced form thiols (R-thiols) and total thiols (T-thiols) without and with Tris-(2-carboxyethyl)-phosphine reduction. UV/HCOOH-induced Hg cold vapor generation was used instead of K(2)SO(8)-KBH(4)/NaOH-HCl and/or KBrO(3)/KBr-KBH(4)/NaOH-HCl systems as an effective interface between RPLC and CVG-AFS. The limits of detection (3sigma) of RPLC-(UV/HCOOH)-CVG-AFS with PHMB labeling for Cys, HCys, Cys-Gly, gamma-Glu-Cys and GSH as well as NAC were 4.6, 5.9, 5.9, 8.1, 7.3 and 5.9nM with the RSD of 4.4, 5.1, 3.6, 7.5 4.2 and 3.7% (n=6 at 2microM), respectively, satisfying the simultaneous determination of the main urinary LMM thiols. This developed method was applied successfully to determine the LMM R-thiols and T-thiols in 10 urine samples contributed by 10 healthy volunteers.

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4-(Hydroxymercuri)benzoic acid sodium salt, ≥95.0% (Hg)