Determination of chlordiazepoxide,diazepam, and their major metabolites in blood or plasma by spectrophotodensitometry

An analytical procedure was developed for the determination of chlordiazepoxide, diazepam and their major metabolites in blood or plasma. Demoxepam, a metabolite of chlordiazepoxide, is determined by spectrofluorometry after selective extraction. The remaining compounds are determined by spectrophotodensitometry after thin-layer chromatographic separation.The sensitivity limit of the spectrofluorometric determinationn of demoxepam is 0.1 to 0.2 μg while that of the spectrophotodensitometric determination of chlordiazepoxide, diazepam and their N-desmethyl metabolites is 0.05 to 0.2 μg. The sensitivity and specificity of the assay renders it suitable for monitoring plasma levels of chlordiazepoxide and its major metabolites following single or chronic oral administration of chlordiazepoxide hydrochloride. The sensitivity limit for diazepam and nordiazepam, its major metabolite, renders the assay useful only for the determination of plasma concentrations resulting from high dosage of diazepam. The assay was used to determine chlordiazepoxide and its metabolites following oral administration of Librium. The data showed a significant correlation to those obtained on the same specimens by differential pulse polarography and by radio-immunoassay.