Novel reductive-reductive mode electrochemical detection of Rohypnol following liquid chromatography and its determination in coffee.

Rohypnol (flunitrazepam) has been successfully determined in coffee by high performance liquid chromatography dual electrode detection (LC-DED) in the dual reductive mode. Initial studies were performed to optimise the chromatographic conditions and these were found to be 50% acetonitrile, 50% 50 mM pH 2.0 phosphate buffer at a flow rate of 0.75 mL min(-1), employing a Hypersil C18, 5 μm, 250 mm × 4.6 mm column. Cyclic voltammetric studies were made to ascertain the redox behaviour of Rohypnol at a glassy carbon electrode over the pH range 2-12. Hydrodynamic voltammetry was used to optimise the applied potential at the generator and detector cells; these were identified to be -2.4 V and +0.8 V for the redox mode and -2.4 V and -0.1 V for the dual reductive mode respectively. A linear range of 0.5-100 μg mL(-1), with a detection limit of 20 ng mL(-1) was obtained for the dual reductive mode. Further studies were then performed to identify the optimum conditions required for the LC-DED determination of Rohypnol in beverage samples. A convenient and rapid method for the determination of Rohypnol in beverage samples was developed using a simple sample pre-treatment procedure. A recovery of 95.5% was achieved for a sample of white coffee fortified at 9.6 μg mL(-1) Rohypnol.