犬组织匀浆中乌头碱的高效液相色谱-质谱测定法及体外稳定性研究(英文)

Determination of aconitine in dog tissue homogenates by HPLC-MS/MS and its application to in vitro metabolic stability study

目的建立了犬组织匀浆中有毒生物碱乌头碱的高效液相色谱-质谱测定法,并应用该方法考察了乌头碱在犬主要组织匀浆中的代谢稳定性。方法色谱柱为C18柱,流动相为含有5 mmol/L甲酸铵和0.2%甲酸的乙腈和水,梯度洗脱。三重四级杆串联质谱配备电喷雾电离源(ESI),正离子模式下以选择离子监测进行检测。乌头碱分别与犬的肝、小肠、胃和肾的组织匀浆温孵,温孵后不同时间点取出,加入内标西酞普兰,乙腈沉淀后取上清液直接进样测定。结果乌头碱浓度在5～500 ng/ml峰面积和内标的比值与浓度呈良好的线性关系;方法的回收率为85.73%～92.12%,其日内、日间的RSD值分别为5.32%～8.95%和5.45%～8.86%。体外孵育2 h后,在犬肝、小肠匀浆中有约20%的乌头碱进行了代谢转化,其t1/2分别为460.6和521.3 min。但在胃和肾中几乎无变化。结论研究提示犬体内的乌头碱主要在小肠、肝脏中代谢转化,但其代谢和清除较慢。

Objective To develop a HPLC-MS/MS method for the determination of aconitine and study the in vitro metabolic stability of aeonitine in dog tissue homogenates. Methods The chromatographic separation was performed on a Cls column. The mo- bile phase consisted of acetonitrile and water with 0. 2% formic acid and 5 mmol/L ammonium acetate. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization interface source was used for the quantitative determination in the positive selective reaction monitor mode. Aconitine was incubated with dog tissue homogenates and samples were withdrawn at different time points and precipitated by acetonitrile with internal standards citalopram. Results Aconitine showed good linear relationship over the range from 5 to 500 ng/ml. The recoveries of aconitine were between 85.73% and 92. 12% at three QC concentration levels. The in- tra- and inter-day precisions were 5.32% -8.95% and 5.45% -8.86%, respectively. After incubation, about 20% of aconitine were cleared in the liver and small intestine, and tl/2 were 460. 6 and 521.3 min, respectively. But none was metabolized in the stom- ach and kidney. Conclusion These results demonstrated that aconitine was mainly metabolized in the liver and small intestine at a slow rate.