LC-MS/MS法测定血浆中山奈酚-3-O-芸香糖苷浓度及其在大鼠药动学研究中的应用  

A new method for determination of kaempferol-3-O-rutinoside in plasma: application to pharmacokinetic study in Sprague-Dawley rats

作　　者：梁世瓢 刘飞[1] 杜飞飞[1] 牛巍[1] 孙艳[1] 李川[1] 郭美丽[2] 

机构地区：[1]中国科学院上海药物研究所药物代谢研究中心,上海201203 [2]第二军医大学,上海201203

出　　处：《中国临床药理学与治疗学》2006年第5期491-496,共6页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：国家"863"计划基金项目资助(№2002AA214051;№2003AA2Z347A);国家"973"计划基金资助(№2005CB523403);上海市基金资助项目(№04DZ19215)

摘　　要：目的：建立能够灵敏、特异、准确、可靠地测定血浆中山奈酚-3-O-芸香糖苷浓度的分析方法。方法：优化山奈酚-3-O-芸香糖苷（kaempferol-3-O-rutinoside，NFR）的离子化及其断裂方式，确定相关液相色谱条件，选择能有效地从血浆样品中提取NFR的样品前处理方法；开展方法学考察，以验证新建分析方法的准确性、精密度等，在此基础上将该方法用于分析大鼠静脉注射NFR后所得的实际血浆样品，以验证新建分析方法的适用性。结果：ESI（＋）为NFR提供最佳的离子化条件，采用SRM工作模式，用m／z595→287来检测NFR。同时，以左旋千金藤啶碱（1-Stepholidine）为内标物化合物（IS），其SRM检测采用m／z328→178。NFR及IS的色谱保留时间（tR）分别为2．3和2．2min。用乙酸乙酯（EtOAc）提取经HCl酸化的大鼠血浆样品，NFR和IS的回收率分别为58．5％～70．1％和72．5％。NFR和IS在整个分析过程中稳定。在0．192～600ng·ml^-1的浓度范围内，对NFR与IS的峰面积比值和NFR的血药浓度进行线性回归，其回归曲线线性良好（r=0．9999，n=6×5）。批内准确性为92％～107％，其精密度为1．0％～5．7％；批间准确性为94％～99％，其精密度为1．5％～8．4％。本方法的LLOQ为0．192ng·ml^-1。大鼠静脉给药单剂量NFR（30 mg·kg^-1）后，NFR的消除半衰期（T1/2）为1．27 h、体内平均滞留时间（MRT）为0．32 h，NFR在大鼠体内的清除率（CL）为2．73 L·h·kg^-1、稳态分布体积（VSS）为0．92 L·kg^-1。结论：应用LC-MS／MS技术建立的测定血浆中山奈酚-3-O-芸香糖苷浓度的新方法灵敏可靠，这项研究工作为全面开展NFR注射液的临床前药代动力学研究打下了重要的分析方法学基础。AIM： To develop a sensitive and reliable method for determination of kaempfeml-3-O-rufinoside （NFR） in plasma. METHODS： The optimal ionization and fragmentation conditions, as well as liquid chromatographic ones, to detect kaempfeml-3-O-rutinoside were identified. Effective sample clean-up method for recovering the analyte from plasma was also studied. The newly developed analytical method was evaluated in terms of accuracy, precison, selectivity, sensitivity, and stability, which was further demonstrated its apphcability in a pilot plasma pharmacokinetics study in Sprague-Dawley rots receiving an intravenous dose of NFR at 30 mg·kg^- 1. RESULTS： Positive ion electrospray ionization was found to effeciently generate precursor protonated molecules of both the analyte and the internal standard （IS, 1-Stepholidine） which was optimized and used to produce their characteristic and abundant fragment ions for MS/MS analysis in selected reaction monitoring mode with precursor-to-product ion transitions m/z 595→287 for NFR and m/z 328 →178 for IS. The chromatographic retention times were 2.3 and 2.2 min for NFR and IS, respectively. Liquidliquid extraction using ethyl acetate provided good recovery for both the analyte and IS from HCl-acidified mt plasma samples demonstrating 58.5 % - 70.1% and72.5 % recovery, respectively. NFR and IS were stable during the whole process of the bioassay. Good linearity was achieved for regresion of NFR/IS peak area ratio to nominal plasma concentration of the analyte demonstrating r value equal to 0.9999 （ n = 6 × 5）, when the concentration was between 0. 192 and 600 ng·ml^-1 . The withinnan acurracy and presicion of the analytical method were quite good for plasma NFR, i.e., 92% - 107% and 1.0% -5.7%, respectively and the between-nm data were 94 % - 99 % and 1.5 % - 8.4 %, respectively. The lower limit of quantification （LLOQ） of the method was 0. 192 ng·ml^-1. Following a single intravenous dose of NFR at 30 mg· kg^-1 to Sprague-Dawley rats, NFR exhibited

关 键 词：山奈酚-3-O-芸香糖苷 液质联用技术 血药浓度 药动学 

分 类 号：R969.1[医药卫生—药理学]