Emara Samy, Masujima Tsutomu, Zarad Walaa, Kamal Maha, Fouad Marwa, El-Bagary Ramzia
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Km 28 Ismailia Road, Cairo, Egypt
P.I. Lab. Single Cell MS, RIKEN Quantitative Biology Center, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan.
J Chromatogr Sci. 2015 Sep;53(8):1353-60. doi: 10.1093/chromsci/bmv024. Epub 2015 Apr 1.
A simple, rapid and environment-friendly direct injection HPLC method for the determination of methyldopa (MTD) in human serum has been developed and validated. The method was based on cleanup and separation of MTD from serum by mixed-mode liquid chromatography using a single protein-coated TSK gel ODS-80 TM analytical column (50 × 4.0 mm i.d., 5 µm). The protein-coated column exhibited excellent resolution, selectivity and functioned in two chromatographic modes: size-exclusion chromatography [i.e., solid-phase extraction (SPE) for serum proteins] and reversed-phase chromatography for the final separation of MTD. SPE and HPLC separation were carried out simultaneously with a green mobile phase consisting of acetate buffer (0.1 M, pH 2.4) at a flow rate of 1 mL/min and at room temperature (23 ± 1°C). The eluent was monitored at emission and excitation wavelengths of 320 and 270 nm, respectively. A calibration curve was linear over the range of 0.1-30 µg/mL with a detection limit of 0.027 µg/mL. This online SPE method was successfully applied to real samples obtained from patients receiving MTD therapy.
已开发并验证了一种简单、快速且环保的直接进样高效液相色谱法，用于测定人血清中的甲基多巴（MTD）。该方法基于使用单一蛋白质包被的TSK凝胶ODS - 80 TM分析柱（50×4.0 mm内径，5 µm）通过混合模式液相色谱从血清中净化和分离MTD。蛋白质包被柱表现出出色的分离度、选择性，并在两种色谱模式下起作用：尺寸排阻色谱法[即用于血清蛋白的固相萃取（SPE）]和反相色谱法用于MTD的最终分离。SPE和HPLC分离同时进行，流动相为绿色，由乙酸盐缓冲液（0.1 M，pH 2.4）组成，流速为1 mL/min，在室温（23±1°C）下进行。分别在发射波长320 nm和激发波长270 nm处监测洗脱液。校准曲线在0.1 - 30 µg/mL范围内呈线性，检测限为0.027 µg/mL。这种在线SPE方法已成功应用于从接受MTD治疗的患者获得的实际样品。
