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In order to establish an analytical method for the determination of phenobarbital (PB), phenytoin (PPH) and their hydroxylated metabolites in hair, animal model experiments were performed. Five male dark-agouti pigmented rats, aged 5 weeks, were intraperitoneally and orally administered PB or PPH independently at 25 mg/kg once a day for 5 successive days. The growing back hair was collected 15d after the first administration. Four typical extraction methods, using NH4OH-methanol-acetone, TFA-methanol-acetone, 1M sodium hydroxide and proteinase K, were evaluated using the rat hair samples containing PB or PPH. Methanol-acetone-NH4OH (10: 10: 1) was the best extraction method from all aspects, such as high extraction efficiency and low noise. The analytes in the extract were methylated in acetonitrile with 20% tetramethylammonium hydroxide and methyliodide at 70 degrees C for 10 min. After purification with Bond Elut Certify, the methylated products were analyzed by GC-MS. From rat hair, PB, p-hydroxy PB, PPH and p-hydroxy PPH were detected at average concentrations of 26.9, trace, 4.2 and 0.4 ng/mg with an intraperitoneal (i.p.) injection, and at 30.9, trace, 4.0 and 0.4 ng/mg with oral administration, respectively. There was little difference in hair concentrations between i.p. injection and oral administration. This method was applied to the head hair of two patients who orally took toxic amounts of PB and two volunteers who orally took 100 mg of PPH daily for 5 d. The hair concentrations of PB in the two patients were 16.2 and 14.7 ng/mg, and those of PPH in the two volunteers were 3.3 and 0.1 ng/mg.

biological and pharmaceutical bulletin

Hair Analysis for Pharmaceutical Drugs. I. Effective Extraction and Determination of Phenobarbital, Phenytoin and Their Major Metabolites in Rat and Human Hair.