Puente Belen, Hernandez Esther, Perez Susana, Pablo Luis, Prieto Esther, Garcia Maria Angeles, Bregante Miguel Angel
Department of Analytical Chemistry, School of Veterinary Science, University of Zaragoza, Spain.
J Chromatogr Sci. 2011 Nov-Dec;49(10):745-52. doi: 10.1093/chrsci/49.10.745.
A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ∼ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.
已开发并验证了一种新的高效液相色谱法，该方法采用柱前衍生化，以金刚烷胺作为内标，用于测定血浆和玻璃体液中青光眼候选治疗药物美金刚。柱前衍生化使用9-芴基甲基氯甲酸酯-氯（FMOC-Cl）作为衍生化试剂，随后用正己烷进行液-液萃取。衍生化的最佳条件为FMOC-Cl浓度1.5 mM、反应时间20分钟、温度30°C、硼酸盐缓冲液pH 8.5以及硼酸盐缓冲液与乙腈的比例为1:1。衍生物通过等度高效液相色谱法进行分析，在Novapack C(18)反相柱上，使用荧光检测器，激发波长λex 260 nm，发射波长λem 315 nm，流动相为乙腈-水（73:27，v/v），温度40°C，流速1.2 mL/min。两种类型的样品线性范围均为10 - 1000 ng/mL，定量限约为10 ng/mL。该分析方法可能适用于眼部可用性研究。
