氨基类神经递质的N-乙基化法UHPLC-MS/MS定量分析技术

N-Ethylation facilitates simultaneous quanti cation of amino neurotransmitters using UHPLC-MS/MS

目的·为了研究氨基类神经递质的含量变化对生物体的生理及病理生理过程的影响,探讨这类代谢物的高灵敏定量分析方法。方法·使用乙醛与氰基硼氢化钠(NaBH_3CN)的一对稳定同位素标记试剂分别对氨基类神经递质进行N-乙基化柱前衍生,采用超高效液相色谱-串联质谱联用技术(ultra-high performance liquid chromatography-tandem mass spectrometry,UHPLC-MS/MS),对20种氨基类神经递质进行一次性高灵敏内标定量分析。结果·方法学验证表明该20种氨基类神经递质均呈现良好的线性关系(相关系数R^2>0.991 0);除组胺检测限为36.5 fmol外,其余物质均在0.5～10 fmol范围;日间差及日内差的相对标准偏差低于15%。使用该方法对人尿液、血清及唾液样本中的氨基类神经递质进行定量检测分析,分别可检测到14、10及8种神经递质。结论·该方法具有较高检测灵敏度及精密度,可用于人尿液、血清及唾液样本中神经递质的定量分析;与已有方法相比,此方法具有反应条件温和、操作简便、反应原料易于淬灭、灵敏度与精密度高等优势,可实现多种氨基类神经递质的一次性高灵敏定量分析。

Objective · To investigate the effect of content changes of neurotransmitters on multiple physiological and pathophysiological processes in mammals, and explore simultaneous sensitive quantification of these metabolites. Methods · An efficient and sensitive method for simultaneous quantification of 20 amino neurotransmitters was achieved through pre-column N-ethylation of neurotransmitters with acetaldehyde and NaBH3CN followed with quantitative analysis using ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）. And NaBH3CN and NaBD3CN were used as a pair of stable isotope labeling reagents together with natural isotopic standards to achieve accurate quantifcation. Results · The analysis showed all 20 neurotransmitters had excellent linearity with R2 over 0.991 0 and sensitivity with the limits of detection （LOD） in the range of 0.5-10 fmol except for histidine with an LOD of 36.5 fmol. The result also showed good robustness with the interday and intraday RSD （relative standard deviation） below 15%. The method was further applied to human urine, serum and saliva samples for validation, in which 14, 10 and 8 neurotransmitters were detected, respectively. Conclusion · The method is of high sensitivity and precision. Compared to previous ones, this method is of great applicability in different matrices, with mild reaction and straightforward procedures. Besides, the reaction reagents are easy to be removed by simple quenching thus avoiding interference in the subsequent analysis.