HPLC-ESI-MS法测定大鼠脑组织中神经递质L/D-丝氨酸的含量

华东师范大学学报(自然科学版) ›› 2013, Vol. 2013 ›› Issue (6): 165-170.

HPLC-ESI-MS法测定大鼠脑组织中神经递质L/D-丝氨酸的含量

王伟莉, 段雅乐, 刘莎, 张耀, 赵政

华东师范大学脑功能基因组学教育部重点实验室，上海市脑功能基因组学重点实验室，药理毒理研究室，上海 200062

收稿日期:2013-01-01 修回日期:2013-04-01 出版日期:2013-11-25 发布日期:2014-01-13

HPLC-ESI-MS determination of neurotransmitters L-serine/D-serine in rat brain tissues

WANG Wei-li, DUAN Ya-le, LIU Sha, ZHANG Yao, ZHAO Zheng

Laboratory of Neuropharmacology and Toxicology, Key Laborarory of Brain Functional Genomics, Ministry of Education, Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai 200062, China

Received:2013-01-01 Revised:2013-04-01 Online:2013-11-25 Published:2014-01-13

摘要/Abstract

摘要： 建立一种检测大鼠脑组织中L-丝氨酸和D-丝氨酸的高效液相色谱—电喷雾离子化—质谱联用技术（HPLC-ESI-MS）的测定方法.大鼠脑组织样品匀浆及离心后，取上清液并加入Marfey试剂对样品中L/D-丝氨酸进行衍生化.HPLC流动相为甲酸铵和乙腈，梯度洗脱；质谱采用电喷雾离子源，在负离子模式下进行定性定量分析.结果显示，L-丝氨酸和D-丝氨酸在22 min内得以分离，在2.5~500 ng/mg和1.2~250 ng/mg浓度范围内分别与各自的色谱峰面积呈良好线性关系；L/D-丝氨酸的最低定量限分别为2.5 ng/mg和1.2 ng/mg，方法回收率均在81%～89%范围内.本法具有省时快速，灵敏度和选择性高等优点.将此法运用于成年和老年大鼠海马及皮层中L-丝氨酸和D-丝氨酸含量测定，结果表明，与成年鼠相比，老年鼠海马和皮层中L-丝氨酸的含量基本不变，而D-丝氨酸含量则明显减少，提示D-丝氨酸的变化与正常的衰老过程密切相关.

关键词: 高效液相色谱—电喷雾离子化—质谱, 脑组织, 神经递质, D-丝氨酸, L-丝氨酸

Abstract: A highly sensitive and selective high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) assay was developed for the determination of neurotransmitters L-serine/D-serine in rat brain tissues. The rat brain was removed, homogenized and centrifuged.L-serine/D-serine in the supernatant was derivatized with the Marfey’s reagent. The derivatives were separated by HPLC under gradient elution of mobile phase consisting of ammonium formate and acetonitrile and detected by ESI-MS in the negative ion mode. Reverse phase chromatographic resolution of the enantiomers was obtained within 22 min. Good linear correlations of the concentration to the peak area were obtained in the ranges of 2.5~500 ng/mg for L-serine and 1.2~250 ng/mg for D-serine. The extraction recoveries for both analysts were in the range of 81% to 89%. The method was then applied to determine the levels of the neurotransmitters in cerebral cortex and hippocampus of the adult and aged rats. The results showed that L-serine levels kept unchanged,while D-serine levels decreased significantly, in cortex and hippocampus of the aged rats compared with those of the adult rats, suggesting an involvement of D-serine in the normal aging process.

Key words: HPLC-ESI-MS, brain tissue, neurotransmitter, D-serine, L-serine

中图分类号:

O657.63

王伟莉, 段雅乐, 刘莎, 张耀, 赵政. HPLC-ESI-MS法测定大鼠脑组织中神经递质L/D-丝氨酸的含量[J]. 华东师范大学学报(自然科学版), 2013, 2013(6): 165-170.

WANG Wei-li, DUAN Ya-le, LIU Sha, ZHANG Yao, ZHAO Zheng. HPLC-ESI-MS determination of neurotransmitters L-serine/D-serine in rat brain tissues[J]. Journal of East China Normal University(Natural Sc, 2013, 2013(6): 165-170.

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