Identification of covalent adducts to protein sulfur nucleophiles by alkaline permethylation

We recently reported on the identification of metabolites of the hepatotoxin bromobenzene covalently bound to rat liver protein sulfur nucleophiles (D. E. Slaughter and R. P. Hanzlik, Chem. Res. Toxicol. 4, 349-359 (1991). Central to that study was our development of a method called alkaline permethylation which converts protein-S adducts of xenobiotic electrophiles to stable extractable thioanisole derivatives. We report here on substantial improvements to our original alkaline permethylation method which should greatly expand its potential utility. Specifically, we have developed significantly milder reaction conditions, eliminated side reactions, improved the amount of and consistency of thioanisole yields from various mercapturic acid model compounds, and increased the overall sensitivity of the method at least 50-fold. Using the procedure described herein it is routinely possible to generate, detect, and identify by GC/MS as little as 2 pmol of a thioanisole derivative. This method is potentially quite general and should prove useful for studies in the toxicology of reactive metabolites, for industrial hygiene and biomonitoring, and for agrichemical residue analysis.