HPLC Analysis of the Isomeric Thioether Metabolites of Styrene Oxide

Abstract

An efficient separation of the isomeric thioether metabolites of styrene oxide was achieved under reversed-phase conditions. The column was eluted isocratically with 15% methanol in buffered solutions of phosphoric acid-tris-hydroxymethylaminomethane. The thioether conjugates were separated by class, and the order of elution was cysteine, cysteinylglycine, glutathione, and N-acetylcysteine. The effect of pH and buffer salt concentration on the HPLC separation was examined. Optimal conditions for a separation were either found at low pH (pH 3 or 4) or neutral pH, both at a high buffer salt concentration (75mM). The positional isomers and stereoisomers comprising each amino acid conjugate sample were separated into two peaks. The variations in k¹ and α observed with changes in pH were interpreted as reflecting the degree of interaction of the ionizable groups in the amino acid residue and the hydrophobic portion of the molecule. This interaction was found to be strongly influenced by the relative stereochemistry of the benzylic carbon center, thus allowing the separation of diastereoisomeric thioethers.