超高效液相色谱-高分辨质谱测定大鼠血清中神经递质及其在电离辐射脑损伤动物模型中的应用

建立了超高效液相色谱-四极杆-静电场轨道阱高分辨质谱（UPLC-Q-Orbitrap HRMS）法快速分析大鼠血清中10种神经递质。以0.1%甲酸-乙腈为沉淀剂，采用蛋白沉淀法进行血清中蛋白质大分子的沉淀和目标分析物的高效萃取。建立目标分析物的精确质量数据库（质荷比、保留时间、同位素峰分布及特征碎片离子）和二级质谱库进行精准匹配鉴定，外标法定量。结果表明，该方法的基质效应为62.4%~112.2%，相对标准偏差（RSD）为1.9%~10.1%；回收率为73.6%~104.2%，RSD为2.6%~10.6%；线性关系良好，相关系数（R²）均大于0.99，检出限及定量限分别为0.05~11.63 nmol/L和0.09~46.50 nmol/L；标准溶液、质控样本及血清样本的稳定性可满足生物样本的分析要求；稀释效应的准确度为5.1%~10.6%，精密度为1.1%~5.3%，均在±15%范围之内；在高浓度样本连续进样3次后分析初始流动相，残留效应为0.4%~4.6%，符合生物样本的分析要求。此外，将本方法应用于电离辐射致脑损伤大鼠血清中神经递质代谢的研究，结果表明，电离辐射导致血清中神经递质代谢紊乱，该结果可为电离辐射损伤的生物标志物筛选提供依据。

Analysis of Neurotransmitters in Rat Serum Using Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry and Its Application to Ionizing Radiation-Induced Brain Injury

A rapid analysis methodology for ten neurotransmitters in serum was developed using ultra performance liquid chromatography-quadrupole-Orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS). A typical protein precipitation (PPT) method was employed for removing protein macromolecular compounds and efficient extraction of the studied compounds. A home-made accurate mass database (mass-to-charge (m/z), retention time, isotopic distribution and characteristic fragment ions) and MS/MS spectra library were used for accurate identification. Quantitative analysis was performed using the extracted m/z of full-scan MS and external standard method. Matrix effect was 62.4%-112.2% and the relative standard deviations (RSDs) were 1.9%-10.1%. The extracted recovery and RSDs were 73.6%-104.2% and 2.6%-10.6%, respectively. Experimental results indicated that good linearities (R²＞0.99) were observed in the respective concentration ranges. The limits of detection (LODs) and limits of quantitation (LOQs) were in the range of 0.05-11.63 nmol/L and 0.09-46.50 nmol/L, respectively. Stability of standard solutions, quality control samples and serum samples can meet the requirements of biological samples analysis. Accuracy of the dilution effect (DE) was 5.1%-10.6% and the precision was 1.1%-5.3%, all within the range of ±15%. Carry-over effect (COE) was 0.4%-4.6% through the analysis of initial mobile phase after three consecutive injections of samples of high concentration. Moreover, this method was applied to the research of neurotransmitters metabolism in the serum of rats with ionizing radiation-induced brain injury. Results demonstrated that ionizing radiation causes neurotransmitter metabolism in serum, and can provide important references for the screening of new biomarkers in serum.