高效液相质谱联用法测定人血清中多奈哌齐和卡巴拉汀药物浓度

Determination of Donepezil and Rivastigmine in Human Serum by High-performance Liquid Chromatography Combined with Mass Spectrum

目的:建立测定人血清中多奈哌齐和卡巴拉汀浓度的方法。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),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,用于多奈哌齐和卡巴拉汀定量的反应离子对分别为m/z 380. 1→m/z91. 1和m/z 251→m/z 206. 5,用于内标定量的反应离子对为m/z 383. 1→m/z 337. 1。结果:两种药物在0. 5～400 ng·ml^(-1)范围内线性关系良好(r> 0. 99),定量下限均为0. 5 ng·ml^(-1);多奈哌齐的日内、日间精密度为2. 06%～12. 51%,相对偏差(RE)为-6. 60%～4. 20%,提取回收率为80. 76%～96. 17%,RSD <15%。卡巴拉汀的日内、日间精密度为1. 69%～9. 31%,RE为-5. 58%～5. 20%,提取回收率为96. 69%～100. 15%,RSD <15%。多奈哌齐和卡巴拉汀血清样品在经历3次冻融(-40℃到室温)循环、-40℃放置75 d条件下均稳定,处理后样品在自动进样器中(4℃)放置5 h均稳定(RSD <15%)。结论:该方法快速、准确、灵敏度高、专属性强,可应用于血药浓度测定和人体药物动力学研究。

Objective： To develop an LC-M/S method for the determination of donepezil and rivastigmine in human serum.Methods： After protein precipitation with 600μl acetonitrile,the serum samples were analyzed by LC-M/S. Using loratadine as the internal standard,a Waters Xselect CSH C_（18）（ 150 mm × 3 mm,2. 5 μm） column was used with the mobile phase consisting of water（ containing 10 mmol·L^-1 ammonium acetate）-acetonitrile（ 20 ∶ 80） at a flow rate of 0. 4 ml·min^-1 with the column temperature at 40 ℃. The ion transitions were performed in a positive electrospray ionization multiple reaction-monitoring mode regarding [M ＋ H]＋as the molecular ion peak of donepezil and rivastigmine monitored with / 380. 1→/ 91. 1 and / 251→/ 206. 5,respectively.The internal standard was monitored with / 383. 1→/ 337. 1. Results： The linear range of donepezil and rivastigmine was 0. 5-400 ng·ml^-1（ r〉0. 99） and the lowest quantification limit was 0. 5 ng·ml^-1. For donepezil,the intra-day and inter-day RSD was2. 06% to 12. 51%,the relative error was-6. 60% to 4. 20%,and the relative recovery was ranged from 80. 76% to 96. 17%（ RSD〈15%）. For rivastigmine,the intra-day and inter-day RSD was 1. 69% to 9. 31%,the relative error was-5. 58% to 5. 20%,and the mean relative recovery was ranged from 96. 69% to 100. 15%（ RSD 15%）. For the two compounds,the serum samples were stable at-40℃ for 75 d and kept stable after three repeated freeze-thaw cycles. The prepared samples were stable in the automatic sample injector（ 4℃） for 5 h（ RSD〈15%）. Conclusion： The developed assay method can be applied in the therapeutic monitoring and pharmacokinetic study of donepezil and rivastigmine in human serum.