The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection.
A determination of glutathione, cysteine, and their disulfides using HPLC and dual electrochemical detection (HPLC-DEC) was described previously but was not validated in biological tissues for these and other important thiols and disulfides (SH/SS). Thus, our objectives were to develop this method to quantify simultaneously reduced and oxidized glutathione, cysteine, cystine, and other SH/SS in various tissues, including human blood and plasma, rat liver and hippocampus, mosquito, and spinach leaf. Optimal conditions were determined for sample processing and analysis using metaphosphoric acid and HPLC-DEC. Authentic standards of 10 common SH/SS compounds were resolved and eluted within 15 min, and all standard curves were linear from 5 to 1600 pmol. Validation was based on the following: First, tissue sample sizes were proportional to peak areas over an eightfold range. Second, recovery of SH/SS added to samples before processing was 96-101%. Finally, the results were equivalent and correlated highly with values for total SH by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) assay (r2 = 0.996) and for total glutathione by DTNB-GSSG reductase assay (r2 = 0.998). The life span of the Au/Hg electrode was limited to 200-500 samples based on the lineal range of standard curves. On the basis of these results, we believe that this method will fill a long-time need for the simultaneous determination of SH/SS in biological tissues.
D000330:Aedes / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003545:Cysteine / Q000032:analysis; D003553:Cystine / Q000032:analysis; D004220:Disulfides / Q000032:analysis*; D004228:Dithionitrobenzoic Acid; D004563:Electrochemistry; D005260:Female; D005978:Glutathione / Q000032:analysis; D006624:Hippocampus / Q000032:analysis; D006801:Humans; D008099:Liver / Q000032:analysis; D010944:Plants / Q000032:analysis; D051381:Rats; D013438:Sulfhydryl Compounds / Q000032:analysis*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D003553:Cystine; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D003545:Cysteine