应用快速分离色谱-串联质谱(RRLC-QQQ)定量分析脑缺血后脑组织中内源性氨基酸的含量

Determination of endogenous amino Acids in brain tissues after cerebral ischemia by RRLC-QQQ

目的:基于RRLC-QQQ建立脑缺血后脑组织中未衍生化的内源性氨基酸的含量测定方法。方法:采用DiamonsilC18(4.6 mm×250 mm,5μm)色谱柱,15 min内同时测定12种氨基酸。乙腈-0.1%甲酸水梯度洗脱,流速为0.5 mL.min-1。离子源为ESI,正离子扫描模式,扫描方式为多反应检测。结果:各氨基酸标准曲线在检测范围内均呈良好线性(r>0.996),其检测限低于1.53μg.L-1,定量限低于3.09μg.L-1,高中低3个浓度水平日内和日间精密度的RSD均小于11%。结论:实际样品测定结果表明与假手术组相比,在脑缺血病理状态下,除N-乙酰天门冬氨酸外,脑组织中其余氨基酸水平均升高。部分氨基酸有显著变化,变化水平在术后12,24 h呈时间依赖性。该方法简便,快速,灵敏。这种方法可以应用于寻找脑缺血生物代谢标记物,探索研究脑缺血分子机制以及中药多成分组合药物协同作用机制。

Objective： To establish a method to determine underivatized endogenous amino acids in brain tissues after cerebral ischemia based on RRLC-QQQ. Method： Diamonsil chromatographic column C18 （4. 6 mm × 250 mm,5 μm） was adopted to determine 12 amino acids in 15 min, with acetonitrile-0. 1% formic acid for gradient elution. The flow rate was set at 0. 5 mL · min^-1. With ESI as the ion source, positive ion scanning mode was adopted for multi-reaction monitoring. Result： Each amino acid standard curve （AAs） showed good linear relationship within the detection range （ r 〉 0. 996 ） , with the limit of detection of less than 11% , the limit of quantitation of less than 3.09 μg · L^-1. The RSD of intra- and inter-day precisions at high, middle and low concentrations were less than 11%. Conclusion ： The determination results of actual samples showed that compared with the levels of AAs of the sham operation group, all of the remaining amino acids apart from N-acetyl-aspartate increased in brain tissues. Some amino acids showed significant changes in a time-dependent manner after the operation. The method is so simple, rapid and sensitive that it can be used for finding bi- ological metabolite markers of cerebral ischemia, and exploring cerebral ischemia molecular mechanisms and synergistic mechanism of combined administration of multi-component traditional Chinese medicines.