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.
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.
Tang
Lu
L
Department of Chemistry & the MOE Key Laboratory of Analytical Science, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China.
Chen
Fan
F
Yang
Limin
L
Wang
Qiuquan
Q
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D005561:Formates / Q000737:chemistry; D006902:Hydroxymercuribenzoates / Q000737:chemistry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D005561:Formates; D006902:Hydroxymercuribenzoates; D013438:Sulfhydryl Compounds; C030544:formic acid; C030601:4-hydroxymercuribenzoate