Honeychurch Kevin C, Smith Gemma C, Hart John P
Centre for Research in Analytical, Materials and Sensors Science, Faculty of Applied Sciences, University of the West of England, Bristol, Frenchay Campus, Coldharbour Lane, Bristol, BS16 1QY, UK.
Anal Chem. 2006 Jan 15;78(2):416-23. doi: 10.1021/ac058035a.
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 x 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 microg 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.
已成功开发出一种采用氧化还原模式下的双电极电化学检测的高效液相色谱法（LC - DED）来测定血清中苯二氮䓬类镇静剂硝西泮。为阐明在玻碳电极上发生的电化学机制，使用1 mM硝西泮溶液在pH值2至12之间、电位范围为 -1.5至 +1.5 V的条件下进行循环伏安法测定。在整个pH范围内观察到两个还原峰；第一个峰标记为R1，与7 - 硝基经4e - 、4H + 还原为羟胺物种一致；第二个更负的峰标记为R2，表明是4 - 5位偶氮甲碱基团经2e - 、2H + 还原的结果。在反向阳极扫描时，观察到一个氧化信号，标记为O1，其被认为是羟胺经2e - 、2H + 氧化为亚硝基的结果。在第二次正向扫描时，观察到一个新的还原峰，标记为R3，其被认为是亚硝基物种还原回羟胺物种的结果。随后开展研究利用羟胺/亚硝基氧化还原对，采用LC - DED检测法测定血清中的硝西泮。发现最佳色谱条件包括含有60%甲醇、40% 50 mM pH 4.1醋酸盐缓冲液的流动相，以及一根Hypersil C18 250 mm×4.6 mm色谱柱。进行了流体动力学伏安法研究以优化双电极检测所需的工作电位。发现“发生器”池的最佳施加电位为 -2.4 V，“检测器”池的最佳施加电位为 +0.5 V。通过对加标牛血清和人血清样品进行多次硝西泮测定来评估所提出的方法。前一项评估在浓度为11.2 μg mL⁻¹下进行，后一项评估在1670 ng mL⁻¹下进行。对于牛血清，硝西泮的回收率为75.8%，相关变异系数为6.
