Speciation and Quantitation of Underivatized and Ellman's Derivatized Biological Thiols and Disulfides by Capillary Electrophoresis

• Published in: Analytical biochemistry 1996-11, Vol.242 (1), p.136-144
• Main Authors: Russell, James, Rabenstein, Dallas L.
• Format: Article
• Language: English
• Subjects: Captopril - analysis; Cysteine - analysis; Disulfides - analysis; Dithionitrobenzoic Acid; Electrophoresis, Capillary - methods; Erythrocytes - chemistry; Glutathione - analysis; Humans; Sensitivity and Specificity; Sulfhydryl Compounds - analysis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1996.0439

Capillary electrophoresis (CE) methods have been developed for the speciation and quantitation of thiols and disulfides of biological interest, including the endogenous compounds glutathione, glutathione disulfide, cysteine, cystine, homocysteine, and homocystine and the therapeutic agents penicillamine, penicillamine disulfide,N-acetylcysteine, and captopril. Good speciation and quantitation were achieved for the underivatized thiols and disulfides using a detection wavelength of 200 nm; detection limits were in the range 20–90 μM(1–4 pmol) using a 50-μm-diameter capillary. To achieve lower detection limits, thiols were derivatized with the thiol-specific probe molecule, 5,5′-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent). Good speciation and quantitation were achieved for the Ellman's derivatized thiols using a detection wavelength of 357 nm; detection limits were in the range 5–50 μM(0.03–0.3 pmol) using a 25-μm-diameter capillary. Both the underivatized and derivatized methods were applied to the determination of glutathione in human erythrocytes. Glutathione concentrations of 2–3 mMwere obtained for the erythrocyte samples analyzed, with good agreement between results obtained by the two methods.