Determination of Clonazepam in Chocolate using High Performance Liquid Chromatography and Further Confirmation by Gas Chromatography-Mass Spectrometry

Clonazepam, a nitrobenzodiazepine and a scheduled drug is one of the highly abused drugs in recent times. An analytical methodology was developed for the detection and quantitation of clonazepam in chocolate sample. The method involves the extraction of the chocolate sample by solvent extraction with chloroform under basic conditions (pH: 8-9). The extract was used for the GC-MS analysis in SIM and full scan modes for the detection of the analyte present. For quantification, the extract was re-dissolved in the mobile phase, Acetonitrile: 40mM potassium di hydrogen-phosphate (6:4,v/v), pH adjusted to 3.9. The separation was achieved on a Phenomenex Gemini C18 HPLC reversed-phase column (150mmx4.6mmx5μ) with the mobile phase and diode array detection with UV range 200-370 nm. The calibration plot for the determination of clonazepam is based on linear regression analysis (y=51.405x + 84.924; r²=0.9989). The limit of detection of clonazepam was estimated as 0.1μg/ml with a signal-to-noise ratio of 3:1 and limit of quantitation of clonazepam was estimated as 0.35μg/ml with a signal-to-noise ratio of 10:1. The clonazepam detected in the chocolate was found to be 0.0763μg/mg of the chocolate. The precision studies given a % RSD range of 0.28-3.9 % with respect to peak area and 0.51-1.03 % with respect to retention time. The method is robust, specific and reliable for the qualitative and quantitative analysis of clonazepam in chocolate and the law enforcement laboratories can utilise the method for routine analysis.