血清中心脏毒素的超高效液相色谱-串联质谱测定法

Determination of cardiotoxin in serum by ultra-high performance liquid chromatography tandem with mass spectrometry

目的建立一种人体血清中心脏毒素的超高效液相色谱串联质谱(UPLC-MS-MS)的检测新方法。方法向100μl血清样品中加入300μl乙腈沉淀蛋白,-20℃冷冻2 h,高速离心10 min,取上层有机相用纯水按照1∶1比例稀释,然后过膜、上机测定。样品分离采用梯度洗脱模式,流动相为0.1%甲酸水溶液-0.1%甲酸乙腈溶液,柱温为70℃,Protein BEH C4色谱柱分离,采用电喷雾离子源(ESI)正离子模式测定血清中心脏毒素浓度,外标法定量分析。结果心脏毒素在2.0~200.0μg/L范围内呈现良好的线性关系,r=0.9992。该方法的回收率为98.6%~115.0%,RSD为3.6%~9.8%;检出限为0.6μg/L。结论该方法操作简单,灵敏度和准确度均较高,适用于血清中心脏毒素的快速测定。

Objective To develop a new method for the determination of cardiotoxin in serum by ultra-high performance liquid chromatography tandem with mass spectrometry(UPLC-MS-MS).Methods Totally 100μl serum sample was precipitated with 300μl acetonitrile for 2 h at-20℃and then separated by high speed centrifugation for 10 min.The upper organic phase was diluted at 1∶1 by pure water,then passed through the membrane,and measured it by UPLC-MS-MS.Separation of the sample was achieved by a Protein BEH C4 column with a gradient elution of 0.1%formic acid aqueous solution-0.1%formic acid acetonitrile solution.The column temperature was 70℃.The concentration of cardiotoxin was measured by electrospray ion source(ESI)with positive ion mode and quantified by external standard method.Results This method showed a good linearity for cardiotoxin in the range of 2.0-200.0μg/L,r=0.9992.The recovery was in the range of 98.6%-115.0%with RSDs of 3.6%-9.8%.The limit of detection was 0.6μg/L.Conclusion This method is simple to operate and has high sensitivity and accuracy,and is applicable to rapid detection of cardiotoxin in the serum.