Determination of flunitrazepam and nitrazepam in beverage samples by liquid chromatography with dual electrode detection using a carbon fibre veil electrode

Abstract

Both nitrazepam and flunitrazepam have been determined by high-performance liquid chromatography dual electrode detection (LC-DED) in the reductive–reductive mode, using a carbon fibre veil electrode (CFVE) in conjugation with a glassy carbon electrode. Initial studies were made to optimise the chromatographic conditions. These were found to be 45% acetonitrile-55% acetate buffer (50 mM, pH 4.1) at a flow rate of 1.0 ml/min, employing a Hypersil C18, 5 μm, 250 mm × 4.6 mm column. Cyclic voltammetric studies performed to ascertain the redox behaviour of nitrazepam and flunitrazepam at a CFVE in the optimised mobile phase. Studies showed that similar voltammetric behaviour was obtained to that report at Hg or glassy carbon based electrodes. Further studies were then carried out to identify the optimum conditions required for the LC-DED determination of nitrazepam and flunitrazepam in beverage samples. Hydrodynamic voltammetry was used to optimise the applied potential at the generator and detector cells; these were identified to be −2.40 and −0.25 V, respectively. A linear range of 2.0 to 100 μg ml⁻¹, with a detection limit of 20 ng ml⁻¹ was obtained. A convenient and rapid method for the determination of both nitrazepam and flunitrazepam in beverage sample was developed. Following a simple sample extraction procedure, extracts were examined using the optimised LC-DED procedure. An average percentage recovery of 95.5% (%CV = 4.5%) for nitrazepam and 78.0% (%CV = 8.8%) was achieved for a sample of “Pepsi Max” spiked at 1.0 μg ml⁻¹ nitrazepam, 1.47 μg ml⁻¹ flunitrazepam.