Determination of Phenytoin and Its Major Metabolites in Human Serum by High-Performance Liquid Chromatography with Fluorescence Detection

• Published in: Analytical Sciences Vol. 15; no. 4; pp. 371 - 375
• Main Authors: HARA, Shuuji, HAGIWARA, Jun, FUKUZAWA, Misa, ONO, Nobufumi, KURODA, Takeshi
• Format: Journal Article
• Language: English
• Published: Tokyo The Japan Society for Analytical Chemistry 01.04.1999; Japan Society for Analytical Chemistry; Japan Science and Technology Agency
• Subjects: Biological and medical sciences; General pharmacology; Medical sciences; Pharmacology. Drug treatments; Physicochemical properties. Structure-activity relationships; Human; Biological fluid; Metabolite; Analytical method; HPLC chromatography; Serum; Phenytoin
• ISSN: 0910-6340; 1348-2246
• DOI: 10.2116/analsci.15.371

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 a reaction 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 by reversed-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.