Measurement of biological thiols and disulfides by high-performance liquid chromatography and electrochemical detection of silver mercaptide formation.
A rapid and sensitive method is described for the measurement of picomole levels of the biological thiols glutathione, cysteine, penicillamine, cysteamine, and ergothioneine by a combination of high-performance liquid chromatography and electrochemical detection (ECD). The compounds were separated isocratically on a reversed-phase C18 column by ion-pair chromatography with a mobile phase containing 5 mM acetic acid and 2.5 mM sodium 1-octanesulfonate. After chromatographic separation, the eluate was combined with silver nitrate dissolved in ammonium nitrate buffer at pH 10.5. A platinum disc electrode was used at -0.1 V vs Ag/AgCl to detect the amount of silver ions that had been consumed by the reaction with thiols. For measurement of disulfide, S-sulfonation with sodium sulfite or electroreduction were used to cleave the disulfide, and the thiol anions produced were detected by HPLC-ECD as for the reduced forms. The method was used to assay thiols and disulfides in biological materials.
Kuninori
T
T
Osaka Women's University, Japan.
Nishiyama
J
J
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004220:Disulfides / Q000032:analysis*; D004563:Electrochemistry; D004912:Erythrocytes / Q000737:chemistry; D005260:Female; D005978:Glutathione / Q000031:analogs & derivatives / Q000032:analysis; D019803:Glutathione Disulfide; D006801:Humans; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D012834:Silver; D013438:Sulfhydryl Compounds / Q000032:analysis*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D012834:Silver; D005978:Glutathione; D019803:Glutathione Disulfide