An improved high-pressure liquid chromatographic assay for secobarbital in serum

A high-pressure liquid chromatographic method for the analysis of secobarbital in serum was developed. Secobarbital was extracted from buffered serum (pH 5.5) with a solvent mix of hexane-ether-n-propanol. 5-(4-Methylphenyl)-5-phenylhydantoin was added as an internal standard. Separation of secobarbital and internal standard from serum constituents and other drugs was achieved on a 5-mum C-18 reversed-phase column using an acetonitrile-phosphate buffer (pH 4.4) mobile phase. The eluent was monitored at 195 nm. The sensitivity limit of the assay was approximately 0.02 microgram/ml with 0.5 ml of serum sample. The application of this method to pharmacokinetic studies in pediatric patients was demonstrated.