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

•This submission describes an unreported electrochemical mechanism for Rohypnol.•This is exploited in a LC-DED method for its determination in Cappuccino.•This results in enhanced sensitivity and selectivity.

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.

Graphical abstractRepresentative chromatograms of “Cappuccino” style white coffee samples obtained by LC-DED in the redox mode for (i) fortified at 9.6 μg mL−1 (ii) LC-DED dual reductive mode, fortified at 9.6 μg mL−1 and (iii) unadulterated. R = Rohypnol.Figure optionsDownload full-size imageDownload as PowerPoint slide