Stereoselective determination of p-hydroxyphenylphenylhydantoin enantiomers in rat liver microsomal incubates by reversed-phase high-performance liquid chromatography using β-cyclodextrin as chiral mobile phase additives

An analytical method was developed for determination of p-hydroxyphenylphenylhydantoin enantiomers in rat liver microsome by using reversed-phase high-performance liquid chromatography. A 50 mm C₈ column was used as the analytical column. The mobile phase was made up of 8.8 mmol/L β-cyclodextrin, 0.25 mol/L urea and 0.05 mol/L ammonium acetate in water. The assay was linear from 2.05 to 410.0 μmol/L for each enantiomer. The limits of detection and of quantitation for the method were 0.90 and 2.05 μmol/L for each enantiomer, respectively. The analytical method afforded average recoveries of 93.59 ± 2.75% and 94.72 ± 1.78% for S- and R-p-hydroxyphenylphenylhydantoin, respectively. The method allowed study of the in vitro glucoronidation of p-hydroxyphenylphenylhydantoin in rat liver microsomal incubates. The stereoselectivity of p-hydroxyphenyl-phenylhydantoin phase II metabolism was observed.