Isolation and identification of benzodiazepine drugs and their metabolites in urine by use of Amberlite XAD-2 resin and thin-layer chromatography.

We used the method described here to detect and identify seven benzodiazepine derivatives--diazepam, chlorodiazepoxide, nitrazepam, cloxazolam, oxazolam, oxazepam, and medazepam--and their metabolites in the urine of rabbits given the seven drugs orally. We column-chromatographed 25-ml samples of urine on Amberlite XAD-2. The drugs and their metabolites in the urine were adsorbed by the resin, irrespective of urinary pH, and upon successive elution with methanol and ethyl acetate/methanol/acetic acid (90/10/0.1 by vol) they could be separated and extracted from the normal components of urine with satisfactory analytical recovery. The conjugated metabolites were then enzymatically hydrolyzed and the hydrolysate was extracted into ethyl acetate and the extract thin-layer chromatographed to detect and identify each drug and each of its metabolites. In an experiment in which the urine of human subjects given 5 mg of nitrazepam orally was analyzed by this method, the metabolites of nitrazepam in the 24-h urine could be identified satisfactorily.