LC-MS/MS法测定人血清中石杉碱甲的浓度

目的:建立以高效液相色谱串联质谱电喷雾检测(LC-MS/MS)法的方法测定人血清中石杉碱甲浓度。方法:以可乐定为内标,待测血清样品经乙酸乙酯液-液萃取,有机相吹干,流动相复溶后进样,色谱柱为Phenomenex Luna CN柱,流动相为甲醇∶0.08%甲酸水溶液(含0.2%乙酸铵)=46∶54。ESI离子源正离子方式检测,选择反应监测(SRM),用于定量的离子分别为石杉碱甲m/z:母离子243.2,子离子210.9,碰撞能30V;内标可乐定m/z:母离子229.9,子离子212.9,碰撞能32V;扫描时间为0.5s。结果:血清中内源性物质不干扰石杉碱甲的检测,石杉碱甲血药浓度在0.08～8ng·mL-1范围内线性关系良好(r=0.9930～0.9960);日内、日间RSD均<15%,低、中、高浓度的平均提取回收率分别为69.9%、63.7%、63.7%。结论:该法操作快速、灵敏、简单、准确,介质用量少,符合生物样品检测要求,可用于临床药动学研究中大批量血样的处理。

Determination of Huperzine A Concentration in Human Plasma by LC-MS/MS

OBJECTIVE： To develop a high performance liquid chromatography-mass spectrography method （LC MS/ MS） for determination of huperzine A in human serum. METHODS： The clonidine was used as the internal standard, and the test serum samples after being liquid- liquid extracted by ethyl acetate, dried in organic phase and reconstituted in mobile phase were analyzed on a Phenomenex Luna CN eolumn interfaced with LC MS with Methanol- 0.08% formic acid （46 ： 54 containing 0.2% ammonium acetate）. Positive ion SRM detection was performed for ESI iron source. Target ions were at m/ z 243. 2→210. 9 for Huperzine A with an impact energy of 30V and m/ z 229. 9→212. 9 for the internal standard with an impact energy of 32V during 0.5 s （scan time） . RESULTS： The assay was proved to be free from the interference from endogenous substance, and the linear concentration range of Huperzine A was 0.08-8 ng · mL ^-1（r = 0.993 0-0.996 0） . The intra- and inter-day precision over the entire concentration range were less than 15%. At low, middle and high concentrations, the mean extraction recoveries of Huperzine A were 69.9%, 63.7% and 63.7%, respectively. CONCLUSION： The LC- MS/MS method is rapid, sensitive, simple, accurate and in which little amount of mediator is needed, thus meeting the requirement for the detection of biological specimen and applicable for the treatment of large batch of serum samples in clinical pharmacokinetic study.