Liquid chromatographic determination of chlordiazepoxide and metabolites in plasma

Sensitive quantitation of chlordiazepoxide and its two principal metabolites, desmethylchlordiazepoxide and demoxepam, in human plasma is achieved by high-pressure liquid chromatography. All three compounds and the internal standard, chlorodesmethyldiazepam, are directly extracted from plasma into benzene:isoamyl alcohol (98.5 : 1.5) at physiologic pH with no sample preparation. After evaporation of the organic solvent, the redissolved residue is chromatographed using a C-18 reverse-phase column. Sensitivity limits are 0.05 microgram of either compound/ml of plasma, with coefficients of variation of less than 8%. The method is applicable to single-dose pharmacokinetic studies of chlordiazepoxide or to monitoring of plasma concentrations during long-term therapy.