改良检测血浆中3种双酯型乌头碱含量的UPLC/Q Exactive MS方法学研究

Study on improved UPLC/Q Exactive MS method for detecting the content of three kinds of diester aconitine in plasma

目的:改良UPLC/Q Exactive MS用于同时测定大鼠血浆中乌头碱、次乌头碱和新乌头碱含量的方法学。方法:采用Dionex UltiMate 3000快速超高效液相色谱仪和Q Exactive四极杆-静电场轨道阱高分辨质谱系统检测。采用HypersilGOLD色谱柱分离,流动相A(0.1%甲酸-水):流动相B(乙腈)=35∶65,通过全扫描/选择离子监测(FullMS/SIM)模式进行定性筛查和定量检测。结果:此检测方法专属性高,绝对基质效应在99.22%~100.79%;线性相关系数r≥0.9995,次乌头碱、乌头碱和新乌头碱在1~64ng/mL范围内线性关系良好,定量下限(S/N=10),次乌头碱为0.2ng/mL,乌头碱和新乌头碱为0.5ng/mL;准确度RE%在-2.3800%~2.131%,精密度RSD%在0.0971%~8.8111%,符合生物样本定量分析要求;稳定性考察结果表明室温放置8h、-20℃冻存2周的条件下稳定,-20℃冻融3次后将不再适合生物样本测定。结论:改良后的方法更适合同时测定大鼠血浆中3种双酯型乌头碱的含量。

Objective:To improve the methodology of UPLC/Q Exactive MS method for simultaneous determination of contents of aconitine(AC),hypaconitine(HA)and mesaconitine(MA)in rat plasma.Methods:The Dionex UltiMate 3000 series high performance liquid chromatograph and Q Exactive quadrupole-electrostatic field orbit trap high resolution mass spectrometry detection system were used for detection.Hypersil GOLD chromatographic column was used to separate.Mobile phase A(0.1% formic acid-water):mobile phase B(acetonitrile)=35∶65.Full mass spectrometry/selective ion monitoring(Full MS/SIM)model was used for qualitative screening and quantitative detection.Results:The detection method is highly specific,with an absolute matrix effect of 99.22% to 100.79%.The linear correlation coefficient r≥0.9995,and AC,HA and MA had a good linear relationship in the range of 1 to 64 ng/mL.According to S/N=10,the lower limit of quantification can be obtained as HA was 0.2 ng/mL,AC and MA were 0.5 ng/mL.The accuracy RE% was-2.3800% to 2.131%,and the precision RSD% was 0.0971% to 8.8111%,which met the quantitative analysis requirements of biological samples.The results of stability investigation showed that it was stable when placed at room temperature for 8 h and at -20℃ for 2 weeks.After freezing and thawing at-20℃ for 3 times,it would no longer be suitable for the determination of biological samples.Conclusion:This new improved method is suitable for simultaneous determination of aconitine,hypaconitine and mesaconitine.