Voltammetric Behavior of Nitrazepam and Its Determination in Serum Using Liquid Chromatography with Redox Mode Dual-Electrode Detection

Abstract

A method involving high-performance liquid chromatography with dual-electrode electrochemical detection in the redox mode (LC-DED) has been successfully developed for the determination of the benzodiazepine tranquilizer, nitrazepam, in serum. To elucidate the electrochemical mechanism occurring at a glassy carbon electrode, cyclic voltammetry was preformed with 1 mM solutions of nitrazepam at pH values between 2 and 12, using a potential range from −1.5 to +1.5 V. Two reduction peaks were observed over the whole pH range; the first, designated R1, was consistent with the 4e^-, 4H⁺ reduction of the 7-nitro group to a hydroxylamine species; the second more negative peak, designated R2, was shown to be the result of a 2e^-, 2H⁺ reduction of the 4−5 azomethine group. On the reverse anodic scan, an oxidation signal was observed, designated O1, which was considered to result from a 2e^-, 2H⁺ oxidation of the hydroxylamine to a nitroso group. On the second forward scan, a new reduction peak, designated R3, was observed, which was considered to result from reduction of the nitroso species back to the hydroxylamine species. Studies were then undertaken to exploit the hydroxylamine/nitroso redox couple using LC-DED detection for the measurement of nitrazepam in serum. The optimal chromatographic conditions were found to comprise a mobile phase containing 60% methanol, 40% 50 mM pH 4.1 acetate buffer, in conjunction with a Hypersil C₁₈ 250 mm × 4.6 mm column. Hydrodynamic voltammetric studies were undertaken to optimize the operating potentials required for dual-electrode detection. It was found that an applied potential of −2.4 V was optimum for the “generator” cell and +0.5 V for the “detector” cell. The proposed method was evaluated by carrying out replicate nitrazepam determinations on spiked bovine and human serum samples. The former evaluation was preformed at a concentration of 11.2 μg mL^-1, and the latter at 1670 ng mL^-1. For bovine serum, the recovery of nitrazepam was found to be 75.8% and the associated coefficient of variation was 6.1% (n = 6). For human serum, the recovery was 74.1% with a coefficient of variation of 7.8% (n = 7). These data suggest that the method holds promise for applications in toxicology and where an alternative reliable method to confirm drug abuse may be required.