Abstract: The candidate drug T-VA (C₂₄H₂₈N₄O₄) was synthesized using two kind of neuroprotective ingredients from Chinese traditional medicinal herbs, and displayed promising protective effect on the injured PC12 cells. In previous study, this beneficial effect was due to the modulation of nuclear transcription factor-κB/p65 (NF-κB/p65) and cyclooxygenase-2 (COX-2) expressions. T-VA also exhibited neuroprotective effect in a rat model of ischemic stroke with concomitant improvement of motor functions. Understanding drug metabolites contributes to discovering and developing the novel drug from the metabolites possessed the pharmacological activities. The structure profile of the metabolites provides an essential perspective for the synthetic refinement and the candidates among an extensive series of potential structures, resulting in an optimum drug effectiveness and safety. Liquid chromatography with electrospray ionization mass spectrometric detection (LC-ESI-MS) has been extensively utilized for the online analysis and structural characterization of the active ingredients and metabolites. Thus, there is a need to determine the primary metabolites and mass fragmentation pathways of T-VA in order to understand its potential pharmacological applications. It is a pathway via LC/LTQ-Orbitrap MS to investigate the mass fragmentation of a candidate drug T-VA and study its metabolites in rats. As a result, a method of LC/MSⁿ was established for the analysis of T-VA and its metabolites in rats. The fragmentation pathway of T-VA was explained using the Analyst V4.0 software. By further analysis of main fragment ions (m/z 317, 285, 135) and structural information (C₁₇H₂₁O₄N₂, RDB:8.5, delta ppm:-3.038 ppm), M1 methyl-3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)-methoxy)benzoate was discovered as one of the main metabolites. According to the suppositional structure of M1, M1-1 was synthesized via condensation reaction, which was determined by nuclear magnetic resonance spectrum (¹H-NMR, ¹³C-NMR) and HRMS. By comparing the mass spectrum characters and chromatographic features of M1-1 and M1, it can confirm the exact structure of M1. Furthermore, the neuroprotective effect of M1 in differentiated PC12 cells were evaluated. As a result, M1 in different concentrations could protect PC12 cells injured by CoCl₂ (EC₅₀=16.01 μmol/L), which was close to T-VA. The result indicated that both T-VA and its main metabolite have neuroprotective effect, which provides references for further new drug design. In this study, metabolite of candidate drug T-VA was obtained by chemical synthesis and verified by MS technique, which provided a novel idea on the study of drug metabolism. M1 may become a potential neuroprotective agent and further studies are currently underway.