High performance liquid chromatographic assay of methylphenobarbital metabolites in urine

A reversed-phase liquid chromatographic procedure was developed for simultaneous quantitation of three metabolites of the anticonvulsant methylphenobarbital in urine. These were p-hydroxyphenobarbital, phenobarbital, and p-hydroxymethylphenobarbital. Enzymatic hydrolysis was employed for liberation of the phenolic barbiturates from their glucuronide conjugates. Two internal standards were used at widely different concentrations, which conferred on the assay an accuracy over a wide concentration range. Concentration and instrument response (ultraviolet absorption at 215 nm) were linearly related over the concentration ranges of interest. The within-batch and between-day coefficients of variation were less than 4% in all cases. Recovery of all three analytes from the urine was nearly complete, and no substances that interfered with the assay were encountered in clinical specimens.