Abstract:Objective: To establish a LC-MS/MS method for simultaneous determination of seven neurotransmitters in mice brain tissue. Method: Mice brain tissue was weighed and homogenized with 4 times the amount of ice-cold 0.2% aqueous formic acid,then homogenates mixed with 0.2% formic acid-acetonitrile for protein precipitation,centrifugation,the supernatant was collected and mixed with internal standard solution,then the mixture was injected into LC-MS/MS system for assay.Chromatographic separation was performed on a TSK Gel amide 80 column (4.5 mm×150 mm,5 μm),column temperature 35℃,mobile phase of acetonitrile-6 mmol·L-1 ammonium formate solution(pH 5.50) (67.5:32.5),flow rate was 0.2 mL·min-1,taking positive ion multiple reaction monitoring(MRM) mode for scanning.Reliability of LC-MS/MS was verified by animal tests,and effect of jujube pulp on mice neurotransmitters levels was investigated. Result: The minimum detection limit of dopamine(DA),epinephrine(E),norepinephrine(NE),5-hydroxytryptamine(5-HT),acetylcholine(Ach),γ-aminobutyric acid(GABA),glutamate(Glu)were less than 0.1 μg·L-1,precision RSD of them were 0.52%,0.71%,0.98%,0.35%,0.32%,0.46%,0.94%,respectively;Accuracy RSD of them were 0.67%,0.46%,0.53%,0.72%,0.48%,0.59%,0.82%,respectively.Compared with the blank group animal,after mice were given donepezil,acetylcholine levels in hippocampus and cortex were significantly increased;While scopolamine had opposite effects after administrated,the content of acetylcholine significantly decreased by comparing with the blank group.After oral administration with jujube pulp,neurotransmitters in mice brain tissue significantly changed,and variation was the same with estazolam. Conclusion: This LC-MS/MS analysis method could be applied to pharmacological mechanisms study of neuropsychiatric drugs.
