HPLC Analysis of the Isomeric Thioether Metabolites of Styrene Oxide

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Abstract
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 Cys, cysteinylglycine, glutathione and N-acetylcysteine. The effect of pH and buffer salt concentration on the HPLC [high-performance liquid chromatography] separation was examined. Optimal conditions for a separation were found at low (pH 3 or 4) or neutral pH, both at a high buffer salt concentration (75 mM). The positional isomers and stereoisomers comprising each amino acid conjugate sample were separated into 2 peaks. The variations in Ki and .alpha. 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 strongly influenced by the relative stereochemistry of the benzylic C center, thus allowing the separation of diastereoisomeric thioethers.

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