LC-MS/MS方法同时测定人血浆和尿液中乌头碱与次乌头碱的含量

Simultaneous Determination of Aconitine and Hypaconitine in Plasma and Urine by LC-MS/MS

目的建立同时测定血浆和尿液中乌头碱(aconitine)与次乌头碱(hypaconitine)含量的LC-MS/MS分析方法。方法采用Phenomenex Gemini C18柱(4.6 mm×150 mm,5μm)为色谱柱,以甲醇-0.1%甲酸(70∶30)为流动相,流速为0.5 mL.min-1,以乙醚提取血浆和尿液中乌头碱与次乌头碱。样品经电喷雾离子源正离子化后,通过API3200三重四级杆串联质谱仪,采用多反应离子监测(MRM)对乌头碱(m/z646.3→105.2)与次乌头碱(616.3→105.2)进行测定。结果生物样品中乌头碱在0.5～40μg.L-1内、次乌头碱在0.25～25μg.L-1内线性关系良好;乌头碱与次乌头碱最低定量限分别为0.5和0.25μg.L-1,在尿液中的平均提取回收率76.54%～81.25%,血浆中的平均提取回收率73.22%～80.31%;日间和日内精密度均小于15%。结论本方法简便、灵敏可靠,适用于血浆、尿液中乌头碱与次乌头碱的分析。

OBJECTIVE To develop a LC-MS/MS method for simultaneous assay of aconitine and hypaconitine in human plasma and urine.METHODS Aconitine and hypaconitine were extracted with diethyl ether from human plasma and urine.The residues were separated on a Phenomenex Gemini C18 column（4.6 mm×150 mm,5 μm） with the mobile phase consisting of methanol-water with 0.1% formic acid（70∶30）.The flow rate was 0.5 mL·min^-1.API3200 triple-stage quadrupole mass spectrometer system equipped with an electrospray ionization source was used as the detector and operated in the positive ion mode.Mutiple reaction monitoring（MRM） using the precursor to product ion combinations of m/z 646.3→105.2 and m/z 616.3→105.2 was performed to detect aconitine and hypaconitine.RESULTS The linear ranges were 0.5-40 μg·L^-1 for aconitine and 0.25-25 μg·L^-1 for hypaconitine.The limits of quantification for aconitine and hypaconitine were found to be 0.5 and 0.25 μg·L^-1,respectively.The average extraction recoveries ranged from 76.54% to 81.25% in urine and 73.22% to 80.31% in plasma.The intra-day and inter-day RSDs were less than 15%.CONCLUSION The method provides a sensitive,accurate,precise and reliable analytical procedure for clinical and toxicological monitoring of aconitine and hypaconitine in human plasma and urine.