Simultaneous determination of five neurotransmitters in neonatal rat hippocampus by adding vitamin C coupled with isotope dilution-ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

Analysis of neurotransmitters in brain tissue is useful for mechanistic studies of the central nervous system. Isotope dilution coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of glutamic acid, γ-amino butyric acid, acetylcholine, dopamine, and serotonin in the hippocampus tissue. A 2% (v/v) acetic acid in water-methanol (9:1, v/v) solution was used to prepare the standards and re-dissolute samples after nitrogen drying. An Ultimate AQ-C18 column (150 mm×4.6 mm, 3 μm) was used as the analytical column and 0.1% (v/v) formic acid in water and methanol were used as the mobile phase. Gradient elution was performed and all target compounds were eluted over 3 min at 28℃. Vitamin C spiked during the sample pretreatment and storage periods significantly retarded the oxidation of dopamine and serotonin and improved the neurotransmitter stability. The developed method showed good linearities (correlation coefficient (R2)>0.998), low detection limits (0.15-1.0 μg/L), good inter-and intra-day precisions (relative standard deviations:0.39%-13.6%), good accuracy (92.9%-119%), low carry-over, and excellent stability. Moreover, the method was successfully applied and validated in the determination of rat hippocampus exposed to bisphenol A.