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

Abstract

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), γ-glutamyl cysteine (γ-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 2SO 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 (3 σ) of RPLC–(UV/HCOOH)–CVG–AFS with PHMB labeling for Cys, HCys, Cys–Gly, γ-Glu–Cys and GSH as well as NAC were 4.6, 5.9, 5.9, 8.1, 7.3 and 5.9 nM with the RSD of 4.4, 5.1, 3.6, 7.5 4.2 and 3.7% ( n = 6 at 2 μM), 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.