Quantitative determination of diazepam, nitrazepam and flunitrazepam in tablets using thin-layer chromatography–densitometry technique

Abstract

A simple and reliable assay for diazepam, nitrazepam and flunitrazepam in tablets by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC)–densitometry is described. A quantity of a ground tablet mass, equal to the average weight of one tablet was sonicated in MeOH, mixed with appropriate internal standard, filtered and either injected directly into the liquid chromatograph, or after evaporation and reconstitution of an aliquot of the extract, was spotted on a silica gel thin-layer plate. A variable UV detector, operated at 254 nm was employed in both procedures. A C18, reversed phase 7 μm column was used for HPLC analysis; the mobile phase was a 1:1 (v/v) mixture of MeOH (40 °C) and 0.01 M phosphate buffer (pH 7, 80 °C). The TLC plate was developed in an unsaturated chromatographic chamber containing 100 ml chloroform–acetone (9:1); at room temperature, the mobile phase was allowed to travel 15 cm. The percentage of the active ingredient content of each tablet obtained by both procedures, was in the range of the stated amount except for one brand of diazepam tablets which contained ∼23% less active ingredient than the minimum prescribed amount. The TLC densitometry, although yields slightly higher values than the HPLC method, is preferred due to its simplicity, ease and low cost.