title: 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).
authors.affiliation: Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Brno, Czech Republic.
authors.forename: Jitka
authors.initials: J
authors.lastname: Petrlova
authors.forename: Radka
authors.initials: R
authors.lastname: Mikelova
authors.forename: Karel
authors.initials: K
authors.lastname: Stejskal
authors.forename: Andrea
authors.initials: A
authors.lastname: Kleckerova
authors.forename: Ondrej
authors.initials: O
authors.lastname: Zitka
authors.forename: Jiri
authors.initials: J
authors.lastname: Petrek
authors.forename: Ladislav
authors.initials: L
authors.lastname: Havel
authors.forename: Josef
authors.initials: J
authors.lastname: Zehnalek
authors.forename: Adam
authors.initials: A
authors.lastname: Vojtech
authors.forename: Libuse
authors.initials: L
authors.lastname: Trnkova
authors.forename: Rene
authors.initials: R
authors.lastname: Kizek
mesh_terms: D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D006801:Humans; D015394:Molecular Structure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D044967:Serum / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000032:analysis*; D003313:Zea mays / Q000737:chemistry
chemical_list: D012997:Solvents; D013438:Sulfhydryl Compounds