Abstract
A highly sensitive fluorometric high-performance liquid chromatographic method was developed for the simultaneous determination of phenytoin and its major metabolites [5-(3-hydroxyphenyl)-5-phenylhydantoin and 5-(4-hydroxyphenyl)- 5-phenylhydantoin]. After extracting these compounds and 5-(4-methylphenyl)-5-phenylhydantoin (MPPH) as an internal standard from serum (50 μl) with ethyl acetate, they were further converted into the corresponding fluorescent derivatives by areaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and dibenzo-18-crown-6 in acetonitrile. The derivatives were separated byreversed-phase chromatography on a YMC-Pack ODS-A column with a mixture of acetonitrile–50 mM phosphate buffer (pH 7.0) (4:6, v/v) as a mobile phase, and were then detected spectrofluorometrically at 448 nm with excitation at 365 nm. The detection limits for phenytoin, 5-(3-hydroxyphenyl)-5-phenylhydantoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin added to serum were 0.6, 3.0 and 0.8 ng (2.4, 11 and 3.1 pmol) ml–1 serum at a signal-to-noise ratio of three. The method was applied to determine the unbound- and total-phenytoin and the metabolites levels in the serum obtained from two healthy volunteers, after oral administration of the drug.
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Hara, S., Hagiwara, J., Fukuzawa, M. et al. Determination of Phenytoin and Its Major Metabolites in Human Serum by High-Performance Liquid Chromatography with Fluorescence Detection. ANAL. SCI. 15, 371–375 (1999). https://doi.org/10.2116/analsci.15.371
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DOI: https://doi.org/10.2116/analsci.15.371