Analysis and quantitation of diazepam in cream biscuits by high-performance thin-layer chromatography and its confirmation by mass spectrometry Original Research Article

An analytical methodology was developed for the detection and quantitation of diazepam in cream biscuits (food matrix constituting high amount of fat, proteins, carbohydrates and minerals) which are used for criminal motives. Extraction was by ethanol and subsequently by a mixture of methanol:acetonitrile:tetrahydrofuran:water (15:55:4:26 v/v). The quantitation (medazepam as internal standard) was carried out by densitometric scanning of the chromatogram at a wavelength of 230 nm. The limit of detection of diazepam in the samples was estimated as 7.5 ng per 6 mm band with a signal-to-noise ratio of 3:1 and limit of quantitation of diazepam in the samples was estimated as 28 ng per 6 mm band with a signal-to-noise ratio of 10:1. The diazepam detected in the biscuit was found to be in the range of 26–41 μg/g. Standard diazepam was spiked to sample biscuits at three levels (100, 500 and 1000 μg/g) and recoveries were found to be 93%. The mean RF values of diazepam and medazepam are 0.6 and 0.47, respectively. The relative standard deviations of diazepam in biscuit samples were found to be 0.06% for RF and 0.62% for peak area within the plate and 0.35% for RF and 2.15% for peak area among the plates. The method is rapid and reliable for qualitative and quantitative analysis of cream biscuits or similar food matrix laced with diazepam and can be used by law enforcement laboratories for routine analysis.