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目的利用直接注入式串联质谱(direct-infusion tandem mass spectrometry,DI-MS/MS)建立一种耗时短、准确度高的同时测定13种氨基酸(amino acids)含量的方法。方法用内标工作液对干血滤纸片中的氨基酸进行提取,取上清液氮气吹干后加入自制的衍生试剂盐酸正丁醇,水浴加热65℃反应20 min,将反应物吹干后用水-乙腈-甲酸(体积比为100∶100∶0.05)的混合溶液复溶,取上清液直接使用串联质谱进行定量分析。使用电喷雾离子源(electrospray ion source,ESI)正离子模式,采用选择性反应监测(selected reaction monitoring,SRM)模式。采用内标法,用峰高加校正因子定量。结果方法的低浓度回收率为85.6%～114.4%、中浓度回收率为92.4%～114.4%、高浓度回收率为88.9%～113.6%,日内和日间变异系数(RSD)分别为2.3%～9.6%、2.5%～10.3%。不同浓度的氨基酸实际浓度与真实浓度之间的相关系数为0.996 1～0.999 7。结论所建立的直接注入式串联质谱法可以同时对干血滤纸片中多种氨基酸进行定量分析。可作为一种与氨基酸代谢相关疾病的检测手段。
Objective To establish a method for the simultaneous determination of thirteen kinds of amino acids by direct injection tandem mass spectrometry (DI-MS / MS). Methods The amino acids in the dry blood filter paper were extracted with internal standard working solution. The supernatant was air-dried with nitrogen and added with n-butanol, a self-made derivatization reagent. The reaction mixture was heated in a water bath at 65 ℃ for 20 min. - acetonitrile - formic acid (volume ratio of 100: 100: 0.05) mixed solution of reconstitution, the supernatant was directly used for quantitative analysis of tandem mass spectrometry. Electrospray ion source (ESI) positive mode was used with selected reaction monitoring (SRM) mode. Using internal standard method, with peak plus correction factor quantitative. Results The recovery rate of low concentration was 85.6% ~ 114.4%, the recovery of middle concentration was 92.4% ~ 114.4%, the recovery of high concentration was 88.9% ~ 113.6%. The intra - and inter - day coefficients of variation (RSDs) were 2.3% 9.6%, 2.5% ~ 10.3%. The correlation coefficient between the actual concentration of amino acids at different concentrations and the true concentration was 0.996 1 ~ 0.999 7. Conclusion The established direct injection tandem mass spectrometry can quantitatively analyze a variety of amino acids in dried blood filter paper at the same time. Can be used as a means of detection of diseases associated with amino acid metabolism.