维生素C抗氧化-同位素内标稀释-超高效液相色谱-串联质谱同时测定新生期大鼠海马体神经递质的含量

脑区神经递质的测定对于研究神经系统的作用机制具有重要意义。该研究建立了同位素内标稀释-超高效液相色谱-串联质谱同时测定双酚A暴露后大鼠海马体中5种神经递质含量的方法,包括谷氨酸、γ-氨基丁酸、乙酰胆碱、多巴胺以及5-羟色胺。选用2%(体积分数)乙酸水-甲醇(9∶1, v/v)溶液配制标准样品及溶解样品,以Ultimate AQ-C₁₈(150 mm×4.6 mm, 3μm)为分离柱,0.1%(体积分数)甲酸水和甲醇为流动相,在28℃柱温下,梯度洗脱,所有化合物在3 min内出峰。添加维生素C能显著减少多巴胺和5-羟色胺在前处理以及样品储存过程中的氧化,极大地提高神经递质的稳定性,从而准确定量。该方法标准曲线线性关系良好,相关系数R^2>0.998,检出限和定量限低,日内和日间精密度为0.39%～13.6%,加标回收率为92.9%～119%,实际进样残留低,已被成功地应用于新生期双酚A暴露后大鼠海马体神经递质含量的测定。

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

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.