Quantitative determination of chlordiazepoxide and its metabolites in serum by fluorescence TLC--densitometry

A sensitive fluorescence TLC--densitometric procedure was developed for the specific determination of chlordiazepoxide (I) and its two metabolites, demoxepam (II) and desmethylchlordiazepoxide (III), in serum. After extraction from serum with ether, I, II, and II were separated by TLC and converted with a sulfuric acid spray to pale blue (Rf 0.63), green (Rf 0.54), and blue (Rf 0.45) fluorescence spots, respectively. Quantitation was accomplished by scanning the plate with a densitometer at 390 (I), 430 (II), and 390 (III) nm. The sensitivities were 0.05 (I), 0.01 (II), and 0.01 (III) microgram/ml of serum. The procedure was successfully applied to measurement of I-III in human serum after oral administration of 20 mg of chlordiazepoxide hydrochloride.