[Chiral separation of racemic 5-(p-hydroxyphenyl)-5-phenylhydantoin by RP-HPLC using eluents containing beta-cyclodextrin]

A chiral RP-HPLC method was developed to assay the 5-(p-hydroxyphenyl)-5-Phenylhydantoin (p-HPPH) enantiomers, the major metabolite of antiepileptic drug phenytoin, in rat hepatic microsomes. A 50 mm FLC-C8 column was used as the analytical column, beta-cyclodextrin (beta-CD) as chiral mobile phase additive and phenobarbital as the internal standard. The detection wavelength was 250 nm. The linear range of p-HPPH enantiomers was 0.5-110 mg/L. The detection limit was 5 ng (S/N = 3). The recoveries of S- and R-p-HPPH were 93.6% +/- 2.8% and 94.7% +/- 1.8% respectively. The RSD within day and between days were less than 2%. The concentration of beta-CD played an important role in separating chiral enantiomers. When the concentration of beta-CD was between 8.8 and 13.2 mmol/L the resolution of p-HPPH enantiomers had the largest value Rs = -1.1. In this work, 8.8 mmol/L beta-CD solution (4 g beta-CD, 6 g urea, and 1.5 g ammonium acetate in 400 mL water) was selected in considering some factors such as column efficiency, solubility of beta-CD etc. Urea can increase the solubility of beta-CD. When urea: beta-CD = 1:1-1.5:1 (g/g), the solubilization of beta-CD was significant. Methanol concentration in mobile phase affected retention time, resolution of p-HPPH enantiomers and solubility of beta-CD.