Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
Talanta. 2010 Jan 15;80(3):1251-6. doi: 10.1016/j.talanta.2009.09.010.
A fully automated, rapid and highly sensitive HPLC method with automated sample pre-treatment by column-switching system and fluorescence detection has been developed for the trace quantitative determination of the new antidepressant reboxetine (RBX) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. Paroxetine (PXT) was used as an internal standard. Plasma samples containing both RBX and PXT, after filtration, were derivatized by heating with NBD-F in borate buffer of pH 8 at 70 degrees C for 30min. The derivatized plasma samples were injected into the HPLC system where an on-line sample clean up was achieved on the pre-treatment column (Co-sense Shim-pack MAYI-ODS) with a washing mobile phase (acetonitrile:2% acetic acid; 40:60, v/v) at a flow rate of 5mLmin(-1) for 1min. After an automated on-line column switching to the analytical Hypersil phenyl 120A column (250mmx4.6mm, 5microm), the separation of the derivatized RBX and PXT was performed using a mobile phase consisting of sodium acetate buffer (pH 3.5):tetrahydrofuran:acetonitrile (55:35:10, v/v/v) at a flow rate of 2.0mLmin(-1). The eluted derivatives were monitored by a fluorescence detector set at an excitation wavelength of 470nm and an emission wavelength of 530nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r=0.9995, n=5) was found between the peak area ratio of RBX to PXT and RBX concentration in the range of 2-500ngmL(-1), with limits of detection and quantification of 0.5 and 1.7ngmL(-1), respectively. The intra- and inter-day precisions were satisfactory; the relative standard deviations were 2.25 and 3.01% for the intra- and inter-day precisions, respectively. The accuracy of the method proved as the mean recovery values were 100.11+/-2.24% and 100.99+/-2.98% for the intra- and inter-day assay runs, respectively. The proposed method involved simple and minimum sample preparation procedure and short run-time (<12min) and therefore it can be applied to the routine therapeutic monitoring and pharmacokinetic studies of RBX.
建立了一种全自动、快速、高灵敏度的 HPLC 方法，采用柱切换系统和荧光检测自动化样品预处理，用于痕量定量测定人血浆中的新型抗抑郁药瑞波西汀（RBX）。采用简单的柱前衍生化方法，用 7-氟-4-硝基苯并-2-氧代-1,3-二唑（NBD-F）试剂。以帕罗西汀（PXT）为内标。经过滤后的含 RBX 和 PXT 的血浆样品，在硼酸盐缓冲液（pH 8）中于 70°C 加热与 NBD-F 衍生化 30min。衍生化的血浆样品以 5mLmin(-1)的流速在线进样到预处理柱（Co-sense Shim-pack MAYI-ODS）上，用洗涤流动相（乙腈：2%乙酸；40:60，v/v）清洗 1min。在自动在线柱切换到分析性 Hypersil 苯基 120A 柱（250mmx4.6mm，5μm）后，用包含乙酸钠缓冲液（pH 3.5）：四氢呋喃：乙腈（55:35:10，v/v/v）的流动相在 2.0mLmin(-1)的流速下实现衍生化 RBX 和 PXT 的分离。洗脱衍生物在激发波长为 470nm 和发射波长为 530nm 的荧光检测器下进行监测。在最佳色谱条件下，RBX 与 PXT 的峰面积比与 RBX 浓度在 2-500ngmL(-1)范围内呈良好的线性关系，相关系数（r=0.9995，n=5）良好，检测限和定量限分别为 0.5 和 1.7ngmL(-1)。日内和日间精密度均令人满意；日内和日间精密度的相对标准偏差分别为 2.25%和 3.01%。方法的准确度证明，内标和日间测定的平均回收率分别为 100.11+/-2.24%和 100.99+/-2.98%。该方法涉及简单且最小的样品制备步骤，运行时间短（<12min），因此可应用于 RBX 的常规治疗监测和药代动力学研究。
