A gas chromatographic/mass spectrometric method for the simultaneous quantitation of 5,5-diphenylhydantoin (phenytoin), its para-hydroxylated metabolite and their stable isotope labelled analogs.

A stable isotope dilution method for the quantitation by GC-MS of 5,5-diphenylhydantoin, its major metabolite (5-(4-hydroxyphenyl)-5-phenylhydantoin), and, simultaneously, their stable isotope labelled analogs (5,5-diphenyl-2-(13)C-1,3-(15)N2-hydantoin and 5-(4-hydroxyphenyl)-5-phenyl-2-(13)C-1,3-(15)N2-hydantoin) is discussed and evaluated. 5,5-di-(pentadeuterophenyl)hydantoin and 5-(4-hydroxy-3,5-dideuterophenyl)-5-phenyl-2-(13)C-1,3(15)N2-hydantoin are used as internal standards. The chemical work-up procedure of serum (1.0 ml) and urine (0.5 ml) involves acid hydrolysis, extraction at pH 7.4 and permethylation of drug and metabolite analogs by extractive methylation. The mass spectrometric technique consists of repetitive scanning over the molecular ion region of the permethylated derivatives of the phenytoin and para-hydroxy metabolite analogs as they elute from the gas chromatograph. Molecular ion abundances are measured. Reproducibility, selectivity and linearity of the method are discussed in the light of the planned applications.