HPLC-MS/MS法测定人血清中美金刚浓度的方法学研究及临床应用

Validation of HPLC-MS/MS for the Determination of Memantine in Human Serum and its Clinical Application

目的:建立测定人血清中美金刚的方法并应用于临床检测。方法:200μl血清样本采用600μl乙腈沉淀蛋白,离心取上清后,采用HPLC-MS/MS测定,以氯雷他定为内标,色谱柱为Waters Xselect CSH C_(18)(150 mm×3 mm,2.5μm),流动相为水(含10 mmol·L^(-1)乙酸铵)-乙腈(20∶80,v/v),流速0.4 ml·min^(-1),柱温40℃,进样量10μl,采用电喷雾离子源,以多反应监测方式进行正离子扫描,以[M+H]^(+)作为分子离子峰,用于美金刚的反应离子对分别为m/z 180.1→m/z 163.1,用于内标定量的反应离子对为m/z 383.1→m/z 337.1,美金刚出峰时间3.72 min,单个样品分析时间4.5 min。结果:美金刚在0.5~400 ng·ml^(-1)范围内线性关系良好(r>0.9993),定量下限均为0.5 ng·ml^(-1);美金刚的日内、日间精密度RSD为1.00%~14.34%,相对偏差为-5.36%~5.33%,提取回收率为67.04%~70.58%,RSD<15%。美金刚样品在经历3次冻融(-40℃到室温)循环、-40℃放置49 d条件下均稳定,处理后样品在自动进样器中(4℃)放置7 h均稳定(RSD<15%)。应用该方法成功测定57例患者血样中美金刚的浓度。结论:该方法快速、准确、灵敏度高、专属性强,可应用于服用美金刚的中重度阿尔茨海默病患者血药浓度测定和人体药物动力学研究。

Objective:To develop a robust,specific and fully validated LC-MS/MS method for the determination of memantine in human serum and apply it to clinical detection.Methods:After protein precipitated by 600μl acetonitrile,the serum samples were analyzed by LC-MS/MS.Using loratadine as the internal standard,a Waters Xselect CSH C_(18)(150 mm×3 mm,2.5μm)column was used with the mobile phase consisting of water(containing 10 mmol·L^(-1) ammonium acetate)-acetonitrile(20∶80,v/v)at a flow rate of 0.4 ml·min-1 and at the column temperature of 40℃.The ion transitions were performed in positive electrospray ionization multiple reaction-monitoring mode regarding[M+H]+as molecular ion peak of memantine and monitoring with m/z 180.1→m/z 163.1,and the internal standard was monitored with m/z 383.1→m/z 337.1.The retention time of memantine was 3.72 min.The total analysis time was 4.5 min.Results:The linear range of memantine was 0.5-400 ng·ml^(-1)(r>0.9993),and the lower limit of quantitation was 0.5 ng·ml^(-1).For memantine,the intra-and inter-day RSDs were from 1.00%to 14.34%,the relative error was from-5.36%to 5.33%,the relative recovery ranged from 67.04%to 70.58%(RSDs<15%).For memantine,the serum samples were stable at-40℃for 49 d and kept stable after three repeated freeze-thaw cycles.The prepared samples were stable in automatic sample injector(4℃)for 7 h(RSDs<15%).The validated method was applied to analyzing 57 memantine-containing samples in a clinical trial and the results were reliable.Conclusion:The developed assay method can be applied to therapeutic drug monitoring and pharmacokinetic study of memantine in human serum.