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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-ruti-noside,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(l-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(30mg·kg-1)后,NFR的消除半衰期(T1/2)为1.27h、体内平均滞留时间(MRT)为0.32h,NFR在大鼠体内的清除率(CL)为2.73L·h·kg-1、稳态分布体积(VSS)为0.92L·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。
Objective: To establish a sensitive, specific, accurate and reliable method for the determination of kaempferol-3-O-rutinoside in plasma. METHODS: The ionization of kaempferol-3-O-ruti-noside (NFR) and its fragmentation patterns were optimized, the relevant liquid chromatographic conditions were determined, and the choice of effective extraction of NFR from plasma samples was performed. The method of sample preparation; conduct methodological investigation to verify the accuracy and precision of the new analytical method. Based on this, the method is used to analyze the actual plasma samples obtained after intravenous injection of NFR in rats to verify the new analysis. The applicability of the method. Results: ESI(+) provides the best ionization conditions for NFR, using SRM mode of operation, and detecting NFR with m/z 595→287. At the same time, L-Stepholidine was used as the internal standard compound (IS). The SRM detection was performed using m/z 328→178. The chromatographic retention times (tR) for NFR and IS were 2.3 and 2.2 min, respectively. The HCl acidified rat plasma samples were extracted with ethyl acetate (EtOAc). The recoveries of NFR and IS were 58.5%-70.1% and 72.5%, respectively. NFR and IS are stable throughout the analysis. In the concentration range of 0.192~600ng·ml-1, linear regression was performed between the peak area ratios of NFR and IS and the blood concentration of NFR, and the regression curve was linear (r=0.9999, n=6×5). The accuracy within the batch ranged from 92% to 107%, with a precision of 1.0% to 5.7%. The accuracy between batches ranged from 94% to 99%, and the precision was 1.5% to 8.4%. The LLOQ of this method is 0.192 ng·ml-1. After intravenous administration of a single dose of NFR (30 mg·kg-1), the elimination half-life (T1/2) of NFR was 1.27 h, the mean in vivo retention time (MRT) was 0.32 h, and the clearance rate of NFR in rats ( CL) was 2.73 L·h·kg-1 and the steady state volume (VSS) was 0.92 L·kg-1. Conclusion: The new method for the determination of kaempferol-3-O-rutinoside in plasma by LC-MS/MS technique is sensitive and reliable. This research has laid the foundation for the comprehensive preclinical pharmacokinetic study of NFR injection. Important analytical methodology foundation.