title: Two-dimensional high-performance liquid chromatographic method to assay p-hydroxyphenylphenylhydantoin enantiomers in biological fluids and stereoselectivity of enzyme induction in phenytoin metabolism.
abstract: A two-dimensional high-performance liquid chromatographic method was developed to assay the enantiomers of a major phenytoin metabolite, p-hydroxyphenylphenylhydantoin (p-HPPH). Racemic p-HPPH was first separated from phenytoin and other interfering peaks by a reversed-phase column and monitored by an ultraviolet detector. At the retention time of p-HPPH, the racemic p-HPPH peak was automatically transferred to a chiral ligand-exchange column to separate R-p-HPPH and S-p-HPPH by a time-programmed column-switching valve. The ratio of enantiomers was measured by a second ultraviolet detector. The method can be used to assay R- and S-p-HPPH enantiomers with reasonable sensitivity and reproducibility. By using this method, the stereoselectivity of enzyme induction and inhibition of phenytoin metabolism was investigated. Male rats were treated with phenobarbital, 3-methylcholanthrene, acetone, Aroclor 1254, pregnenolone-16 alpha-carbonitrile, dexamethasone and isosafrole. Microsomes were prepared from the rat liver and phenytoin hydroxylation was measured. Pretreatment with phenobarbital, pregnenolone-16 alpha-carbonitrile or acetone induced phenytoin metabolism non-stereoselectively. Pretreatment with dexamethasone decreased R-p-HPPH formation without affecting the formation of S-p-HPPH. Liver microsomes from female rats showed a higher S-p-HPPH formation, whereas R-p-HPPH formation remained the same. Various inhibitors were added to inhibit phenytoin metabolism by control microsomes. Sulphaphenazole, ketoconazole, 4,4-di(p-methoxyphenyl)hydantoin, cimetidine and diazepam inhibited the formation of R- and S-p-HPPH. Quinidine, tolbutamide and mephenytoin showed no significant inhibitory activity. None of these inhibitors showed stereoselectivity.
authors.affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: C Y
authors.initials: CY
authors.lastname: Hsieh
authors.forename: J D
authors.initials: JD
authors.lastname: Huang
mesh_terms: D000096:Acetone / Q000494:pharmacology; D000818:Animals; D001140:Aroclors / Q000494:pharmacology; D020111:Chlorodiphenyl (54% Chlorine); D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002927:Cimetidine / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D003975:Diazepam / Q000494:pharmacology; D004790:Enzyme Induction; D005260:Female; D007654:Ketoconazole / Q000494:pharmacology; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D010672:Phenytoin / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D012451:Safrole / Q000494:pharmacology; D012727:Sex Characteristics; D013237:Stereoisomerism; D013426:Sulfaphenazole / Q000494:pharmacology
chemical_list: D001140:Aroclors; D013426:Sulfaphenazole; D020111:Chlorodiphenyl (54% Chlorine); D000096:Acetone; C027364:5-(3-hydroxyphenyl)-5-phenylhydantoin; D008748:Methylcholanthrene; D010672:Phenytoin; D003907:Dexamethasone; D002927:Cimetidine; D003975:Diazepam; D007654:Ketoconazole; D012451:Safrole; C015959:isosafrole; D010634:Phenobarbital