作者：付晓芸¹, 唐雪², 李星影¹, 毕禄莎¹, 孙嘉茵¹, 徐小平¹

作者单位：1. 四川大学华西药学院, 四川 成都 610041;
2. 岛津企业管理(中国)有限公司成都分析中心, 四川 成都 610063

关键词：氨基酸类神经递质;三叉神经;超高效液相色谱;三重四极杆质谱

摘要：

该文采用UHPLC-MS/MS建立一种同时测定大鼠三叉神经节中4种神经递质的方法。SD大鼠三叉神经节组织匀浆后，加乙腈沉淀蛋白，制备成供试品溶液；以Discovery HS F5-3（2.1 mm×150 mm，3.0 μm）为色谱柱，0.1%甲酸溶液（A相）和0.1%甲酸-40%乙腈溶液（B相）为流动相，梯度洗脱进行分离；然后在+ESI和MRM模式下检测，以Glycine-N¹⁵为内标。结果表明：4种氨基酸的线性关系良好（r²≥0.999），检测限和定量限分别为0.14~1.23 ng/mL和0.47~4.11 ng/mL；在浓度范围内，进样精密度、日内和日间精密度良好，RSD分别在0.90%~5.17%，3.01%~7.19%，4.64%~14.49%；加样回收率为94.8%~108%。大鼠三叉神经节炎症模型在0，1，3，5，7 d 4种神经递质的含量均发生不同程度的变化，其中天门冬氨酸（Asp）在炎症期没有明显的变化（P>0.05），而其余3种递质变化显著（P<0.05），且谷氨酸（Glu）最明显，体外实验也证实 Glu 在炎症条件下含量增加。所建方法可高效同时分离检测三叉神经中4种氨基酸的含量，有较好的使用价值。

Determination of trigeminal neurotransmitters by UHPLC-MS/MS
FU Xiaoyun¹, TANG Xue², LI Xingying¹, BI Lusha¹, SUN Jiayin¹, XU Xiaoping¹
1. West China School of Pharmacy, Sichuan University, Chengdu 610041, China;
2. Shimadzu (China) Co., Ltd., Chengdu Analysis Center, Chengdu 610063, China
Abstract: To establish a method for the simultaneous determination of four amino acid neurotransmitters in rat trigeminal ganglion by UHPLC-MS/MS. After taking the homogenate of the trigeminal ganglion tissue of SD rats, the protein was precipitated with acetonitrile to prepare the test solution. In Discovery HS F5-3 (2.1 mm&#215;150 mm, 3.0 μm) column with 0.1% formic acid solution phase A and 0.1% formic acid-40% acetonitrile solution phase B as mobile phase, gradient elution was used to separate the samples, and then were detected by ESI and MRM. Internal standard method was used for determination with Glycine-N¹⁵ as internal standard. The linearity of four amino acids is good (r² ≥ 0.999). The detection limit and quantitative limit are 0.14 ng/mL-1.23 ng/mL and 0.47 ng/mL-4.11 ng/mL, respectively. The range of concentration, the injection precision, intra-day and inter-day precision were 0.90%-5.17%, 3.01%-7.19%, 4.64%-14.49%, respectively. The recoveries were 94.8%-108%. The contents of four amino acid neurotransmitters in rat trigeminal ganglia inflammatory model changed in varying degrees at 0, 1, 3, 5, and 7 days. Asp had no significant change in inflammatory phase (P>0.05). The other three transmitters changed significantly (P<0.05) and Glu was the most obvious. In vitro experiments also confirmed that the content of Glu increased under inflammatory conditions. The established method can be used for simultaneous separation and detection of four amino acids in trigeminal nerve. It is a good analytical method for detecting neurotransmitters in the mechanism of trigeminal nerve pain.
Keywords: amino acid neurotransmitter;trigeminal;ultra-high performance liquid chromatography;triple quadrupole mass spectrometry
2019, 45(1):64-69  收稿日期: 2018-06-15;收到修改稿日期: 2018-07-26
基金项目:
作者简介: 付晓芸(1995-),女,宁夏银川市人,硕士研究生,专业方向为药物分析
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