Mass screening and confirmation of diazepam in urine by EMIT-thin-layer chromatography

A rapid, sensitive, and specific procedure is described for the mass screening and confirmation of diazepam (Valium) in urine at levels as low as 0.5 micrograms/mL. Raw urine specimens are screened directly by the enzyme multiplied immunoassay technique, and those screened positive are confirmed by thin-layer chromatography. Diazepam and its metabolites are converted to benzophenones; diazepam, 3-hydroxydiazepam, and its glucuronide yield 2-methylamino-5-chlorobenzophenone (MACB); whereas N-desmethyldiazepam, oxazepam, and oxazepam glucuronide yield 2-amino-5-chlorobenzophenone (ACB). The two benzophenones are well separated by a solvent system of petroleum ether:glacial acetic acid (90:10) and can be detected under ultraviolet light. A specimen reported positive for diazepam must produce spots for both ACB and MACB. No other benzodiazepines used in the United States or other urinary substances have been found to interfere; thus the procedure described is specific for for diazepam.