The High-Pressure Liquid Chromatographic Determination of Chlordiazepoxide and Its N-Demethyl Metabolite in Mouse Brain

Abstract This report presents a simple and accurate method for the analyses of chlordiazepoxide and its N-demethyl metabolite in small tissue samples such as mouse brains. The procedure involves the addition of diazepam as the internal standard, homogenization of the mixture with 0.01 N NaOH, and extraction with heptane containing 1.5% (v/v) iso-amyl alcohol. After evaporation of the organic solvent, the residue is dissolved in methanol and the compounds separated by reverse phase high pressure liquid chromatography with 66% (v/v) methanol in water as eluant in isocratic conditions. The analysis is linear for concentrations ranging from 0.5 to 50 ng/mg brain for chlordiazepoxide and the metabolite. The method was applied to the study of the distribution of chlordiazepoxide and the N-demethyl metabolite to the brain of mice receiving intraperitoneally 10 mg/kg chlordiazepoxide HCl.