Abstract
A sensitive thin‐layer chromatographic method is described for the determination of the 7–amino‐metabolites of flunitrazepam and clonazepam. The same procedure is applicable to other 7‐nitro‐benzodiazepines such as nitrazepam. A purified plasma extract of the 7‐amino‐metabolites is separated by thin‐layer chromatography. The primary aromatic amines are diazotized and coupled with N‐(1‐naphthyl) ethylene‐diamine on the thin‐layer plate. The quantities of the 7‐amino‐metabolites are directly evaluated by colorimetric densitometry. The lower limit of detection is in order of 0.5–10 ng cm^−3^ of plasma. The relative standard deviation of the whole procedure is less than ±15% in the range of 0.5–10 ng cm^−3^ for a single determination.
The unchanged drugs can be reduced on the thin‐layer plate and detected by the same method. Since this procedure is rather difficult to perform, it is advantageous to determine the 7‐nitro‐benzodiazepines in plasma by gas chromatography.
The thin‐layer chromatographic method was used to measure the 7‐amino‐metabolites in plasma of patients on either a flunitrazepam or a clonazepam oral dosing regimen.