Simplified method for simultaneous determination of diazepam and its metabolites in urine by thin-layer chromatography and direct densitometry.

A direct densitometric method for determination of diazepam and its metabolites in urine was developed. The proposed procedure involves acid hydrolysis of urine specimens, thereby converting diazepam and its metabolites into benzophenones [2-methylamino-5-chlorobenzophenone (MACB) and 2-amino-5-chlorobenzophenone (ACB)]. It is followed by extraction with chloroform-isopropanol (3:1, v/v). The two benzophenones were separated on thin-layer chromatography plates using hexane-diethyl ether-acetic acid (80:10:10) as a mobile phase. Quantitation of the MACB and ACB spots was achieved by direct ultraviolet densitometry. The limit of detection was 0.5 micrograms per ml of urine for both benzophenones. The proposed method is simple, rapid, reproducible and has been found to be effective for direct determination of diazepam and its metabolites in urine.