LC-MS/MS法测定环鸟苷酸-腺苷酸的方法学建立及其应用

Development and application of an LC-MS/MS method for the determination of cyclic GMP-AMP

目的建立LC-MS/MS方法检测环鸟苷酸-腺苷酸(cGAMP)含量。方法选用程序梯度法,色谱柱为PC HILLIC色谱柱(2.0 mm×150.0 mm,5μm),流动相A为10 mmol/L甲酸铵水溶液(含0.1%甲酸),流动相B为0.1%甲酸乙腈溶液,流速为0.4 ml/min,柱温为30℃,进样量为10μl。质谱优化条件:多反应监测(mμltiple reaction monitoring,MRM)模式,cGAMP[M+H]+为675.22,锥孔电压56 V,碰撞能量为20 eV。结果cGAMP在1~1000 ng/ml浓度范围内线性关系良好(n=9,r2=0.9999);方法定量限为1 ng/ml,批间/批内精密度均<20%,批间准确度为83%~96%,批内准确度87%~95%;方法提取回收率为80%~90%,基质效应<10%。结论该检测方法简单、快速、灵敏、准确,适用于细胞或体外cGAS酶合成cGAMP定量分析。

Objective To establish an LC-MS/MS method for the determination of cyclic GMP-AMP(cGAMP).Methods cGAMP was determined by program gradient method using the PC HILLIC column(2.0 mm×150.0 mm,5μm).The mobile phase A was 0.1%HCOOH aqueous solution containing 10 mmol/L HCOOHNH4,the mobile phase B was 0.1%HCOOH acetonitrile solution and the flow rate was 0.4 ml/min.The column temperature was 30℃and the injection volume was 10μl.Multiple reaction monitoring(MRM)was employed to select cGAMP at m/z 675.22.The cone hole voltage was 56 V and the collision energy was 20 eV.Results The calibration curve showed good linearity ranging from 1 to 1000 ng/ml(n=9,r2=0.9999).The lower limit of quantification was 1 ng/ml.The intra-day and inter-day precisions of cGAMP were<20%,with the accuracy of 83%~97%.The matrix effect was<10%,with the extraction recovery of 80%-90%.Conclusion The established LC-MS/MS method shows good sensitivity and accuracy,which can be used for the determination of cGAMP synthesized via enzymatic synthesis with the cyclic GMP-AMP synthase(cGAS)in cells or in vitro.