ANALYSIS OF GLUTATHIONE, GLUTATHIONE DISULFIDE, CYSTEINE, HOMOCYSTEINE, AND OTHER BIOLOGICAL THIOLS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY FOLLOWING DERIVATIZATION BY N-(1-PYRENYL)MALEIMIDE

The compound N-(1-pyrenyl)maleimide (NPM) reacts with free sulfhydryl groups to form fluorescent derivatives. A new method for measurement o f glutathione and other biological thiols utilizing reverse-phase high -performance liquid chromatography to separate and quantify these deri vatives is described. Separation and quantification of glutathione, cy steine, homocysteine, cysteinylglycine, and gamma-glutamylcysteine der ivatives are achieved. The method allows for the measurement of glutat hione disulfide by masking free glutathione with 2-vinylpyridine, redu cing glutathione disulfide with glutathione reductase, and measuring t he resulting glutathione. Coefficient of variations for the various th iols measured by the NPM method range from 1.5 to 8.8%. The lower dete ction limit is around 50 fmol of glutathione, NPM derivatives are show n to be stable for 2 months at 4 degrees C. Between 94.2 and 97.2% of glutathione and/or glutathione disulfide added to a sample is recovere d using the NPM method. The NPM method is compared to the monobromobim ane highperformance liquid chromatography method and the Tietze assay by measuring glutathione in homogenates from five different cell lines . The newly developed method offers some advantages over the currently accepted techniques, including specificity, speed, sensitivity, and e ase of use. (C) 1995 Academic Press, Inc.