Stereoselective determination of p ‐hydroxyphenyl‐phenylhydantoin 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 C 8 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 glucuronidation of p ‐hydroxyphenylphenylhydantoin in rat liver microsomal incubates. The stereoselectivity of p ‐hydroxyphenylphenylhydantoin phase II metabolism was observed. Copyright © 2001 John Wiley & Sons, Ltd.