固相萃取-高效液相色谱-电喷雾串联质谱技术分析大鼠血浆中的氨基酸及其在电离辐射损伤动物模型中的应用

建立了基于高效液相色谱-电喷雾串联质谱(HPLC-ESI MS/MS)与固相萃取(SPE)相结合的大鼠血浆中氨基酸的分析方法。以硅胶基质的强阳离子交换固相萃取柱(SCX-SPE)提取血浆中的氨基酸和降低基质效应,HPLC-ESI MS/MS进行测定。对SPE处理的样品pH值进行了优化,发现pH 2.8时SCX-SPE提取氨基酸的回收率和重现性比较令人满意。除赖氨酸和鸟氨酸外,氨基酸的总体回收率在33.6%～107.7%之间;除精氨酸外,氨基酸标准曲线的线性相关系数r2＞0.99; 25种氨基酸测定的日内精密度和日间精密度较好,相对标准偏差(RSD)分别低于9.0%和19.1%。此外,将该分析方法用于电离辐射对大鼠血浆氨基酸代谢的影响研究。结果表明,辐射可导致血浆中氨基酸代谢紊乱,并且其紊乱程度与电离辐射损伤程度正相关。研究结果为筛选新的急性辐射损伤标记物研究提供了实验依据。

Analysis of amino acids in rat plasma by solid phase extraction-high performance liquid chromatography-electrospray tandem mass spectrometry and the application to radiation injury rat model

A robust and sensitive high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI MS/MS) method was developed with solid phase extraction (SPE) sample preparation. It was optimized for the analysis of various amino acids in rat plasma. Silica-based strong cationic exchange SPE cartridges were used to extract amino acids and minimize the ion-suppression effect. The SPE cleanup protocol was optimized and it was found that the sample loading pH was very critical for the recovery and reproducibility. When the plasma sample was mixed with 0.1 mol/L acetic acid (pH 2.8) and loaded on SPE cartridge, the recoveries and repeatability for most of the amino acids were satisfactory. The method was fully validated. The analytical characteristics such as total recoveries (33.6%~107.7%, except lysine and ornithine), linearities (r2＞0.99 except arginine), intra-day precisions (relative standard deviation (RSD)＜9.0%) and inter-day precisions (RSD＜19.1%) were satisfactory for most of the amino acids. Furthermore, the method was successfully applied to study the ionizing radiation exposure induced changes in amino acid concentration of plasma samples from rats. Experimental results showed that the ionization radiation could lead to metabolism disorder of plasma amino acids, and the degree of disorder was related with the degree of injury by the ionization radiation injury. Some of the amino acids may be potential biomarkers of ionization radiation injury. These results provide experimental basis for screening of new markers of the acute radiation damage.