Fluorescence--TLC densitometric determination of diazepam and other 1,4-benzodiazepines in serum

A sensitive fluorescence--TLC densitometric procedure was developed for the specific determination of diazepam (I) and its two metabolites, desmethyldiazepam (II) and oxazepam (III), in serum. After extraction from serum with benzene, the compounds were separated by TLC and converted with a sulfuric acid spray to greenish-yellow fluorescence spots with Rf values of 0.72, 0.43, and 0.17, respectively. Quantitation of the TLC plate was accomplished by scanning with a densitometer at 380 nm. The sensitivities of the assay were 18 (I), 6 (II), and 5 (III) ng/ml of serum. This procedure was also was applicable to other 1,4-benzodiazepines in biological fluids.