Simultaneous determination of eight biologically active thiol compounds using gradient elution‐liquid chromatography with Coul‐Array detection

Abstract

The most active form of sulfur in biomolecules is the thiol group, present in a number of biologically active compounds. Here we present a comprehensive study of thiol analysis using flow injection analysis/HPLC with electrochemical detection. The effect of different potentials of working electrodes, of organic solvent contents in the mobile phase, and of isocratic and gradient elution on simultaneous determination of thiol compounds (cysteine, cystine, N-acetylcysteine, homocysteine, reduced and oxidised glutathione, desglycinephytochelatin, and phytochelatins) are described and discussed. These thiol compounds were well separated and detected under optimised HPLC-electrochemical detection conditions (mobile phase: 80 mM trifluoroacetic acid and methanol with a gradient profile starting at 97:3 (TFA:methanol), kept constant for the first 8 min, then decreasing to 85:15 during one minute, kept constant for 8 min, and finally increasing linearly up to 97:3 from 17 to 18 min; the flow rate was 0.8 mL/min, column and detector temperature 25 degrees C, and the electrode potential 900 mV). We were able to determine tens of femtomoles (3 S/N) of the thiols per injection (5 microL), except for phytochelatin5 whose detection limit was 2.1 pmole. This technique was consequently used for simultaneous determination of compounds of interest in biological samples (maize tissue and human blood serum).

Caveolin-1 as a potential high-risk prostate cancer biomarker

“…GSH concentration was calculated from a calibration curve (0.5-100 µM). The signal was quantified as a sum of current responses from all working electrodes (54,55). In the case of real sample measurements, the shift of the retention time was of about ±2%.…”

A rapid LC-FTMS method for the analysis of cysteine, cystine and cysteine/cystine steady-state redox potential in human plasma

“…GSH, GSSG, Cys, CySS, iodoacetic acid (IAA), [1,[2][3][4][5][6][7][8][9][10][11][12][13] C]-IAA, acetonitrile, HPLC grade water, formic acid (98%) and ammonium bicarbonate were purchased from Sigma Chemical (St. Louis, MO). IAA was recrystallized twice in petroleum ether.…”

“…Routine clinical determination utilizes oxidation to cysteic acid [9] to measure total Cys and does not provide information about the steady-state redox potential. Most methods that distinguish Cys and CySS rely upon HPLC with either spectrophotometric [10], fluorometric [11,12] or electrochemical detection [13]. The spectrophotometric and fluorometric assays using reagents targeting thiols depend upon two analyses, one with reduction for detection of disulfides, while assays with reagents targeting amines allow detection of both Cys and CySS directly.…”

Redox status expressed as GSH:GSSG ratio as a marker for oxidative stress in paediatric tumour patients

“…For many years, GSH has been measured by several analytical methods. In particular, high performance liquid chromatography (HPLC) with various detection techniques including ultraviolet (UV) absorbance and fluorescence detection, mass spectrometry and/or electrochemical detection (ED) are commonly used for determination of GSH and GSSG concentrations (46)(47)(48)(49). Each method has its advantages and limitations and may serve a particular need in analysis (50).…”