UPLC/Q Exactive MS快速测定食蟹猴体内黄藤素的血药浓度

Rapid Determination of Palmatine in Cynomolgus Monkey Plasma by UPLC/Q Exactive MS

目的建立UPLC-Q Exactive-MS快速检测食蟹猴血浆中黄藤素的方法。方法采用Ulti Mate 3000系列高效液相色谱仪和Q Exactive四极杆-静电场轨道阱高分辨质谱系统检测。猴含药血浆采用乙腈沉淀蛋白,盐酸小檗碱为内标,蛋白沉淀后用乙腈稀释并测定,色谱柱为Hypersil GOLD Dim C18 column (100 mm×2.1 mm,1.9μm),乙腈-0.1%甲酸水为流动相,梯度洗脱,流速0.3m L/min,柱温30℃,电喷雾正离子化(ESI+),选择性离子扫描模式(t-SIM):黄藤素,[M-Cl]+,m/z 352.15433;内标盐酸小檗碱,[M-Cl]+,m/z 336.123 03。分辨率70000,自动增益控制(AGC)和离子注入时间(IT)分别设为5e4和200 ms;isolation window设为4.0 m/z。喷雾气为氮气,碰撞气为高纯氮气。结果黄藤素在105.0~8 200μg/L (进样样品溶液浓度为0.262 5~20.50μg/L)浓度范围内线性关系良好(线性方程为Y=0.001 188 51C-0.000 042 206 6,r=0.999 6,权重1/C2),最低定量限为105.0μg/L,日内与日间精密度均小于4.06%,目标物和内标基质效应因子为91.71~97.74%(n=5),未处理血浆样品于室温放置约1 d、-20℃放置1 d/10 d/35 d、-20℃放置35 d后室温冻融2次稳定性良好。结论建立的LC-MS分析检测方法线性范围宽、分析时间短、准确度好、灵敏度高、特异性强,适于黄藤素药代及毒代动力学分析检测。

Objective To develop a UPLC-Q Exactive-MS method for the rapid determination of palmatine in cynomolgus monkey plasma.Method It was detected by using an UltiMate 3000 high-performance liquid chromatography-tandem mass spectrometry system with Q Exactive quadrupole-electrostatic field orbitrap.The monkey plasma was precipitated and diluted with acetonitrile containing berberine to act as an IS.The analytical column was packed with accucore Hypersil GOLD Dim C18(100 mm×2.1 mm,1.9μm).The mobile phase was acetonitrile and water(0.1%formic acid)at gradient elution and a flow rate of 0.3 mL/min.The column temperature was 30℃.The ESI sources were operated in the positive ion mode(ESI+).The scan type for MS detection was set to slective ion scan mode(t-SIM)The details of parameters was shown below:palmatine,[M-Cl]+,m/z 352.15433;berberine,[M-Cl]+,m/z 336.12303,resolution 70000,automatic gain control(AGC)And ion implantation time(IT)was set to 5e4 and 200 ms respectively.The spray gas and collision gas was nitrogen and high purity nitrogen.Results Excellent liner relationship was obtained from the range of 105.0~8200μg/L(concentration of injection solution 0.2625~20.50μg/L,linear equation is Y=0.00118851C-0.0000422066,r=0.9996,weight 1/C2),and the limit determination of PMH was 105.0μg/L.The inter-and intra-day RSD was less than 4.06%.The matrix effector of inter-and intra-day was 91.71~97.74%(n=5).Dosing plasma was stable under the follwing conditions:room temperature for about 1 day,-20℃for 1 day/10 days/35 days,and-20℃for 35 days,the room temperature freeze-thaw twice.Conclusion The method with wide linear range proved accurate,simple and rapid with high sensitivity and specificity applied to the analysis of the palmitine in pharmacokinetics and toxicokinetics.