Abstract: 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 sisotope labeling reagents together with natural isotopic standards to achieve accurate quantification. 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 removedsimple quenching thus avoiding interference in the subsequent analysis.

Key words: amino-group, neurotransmitters, pre-column N-ethylation, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), quantitative analysis