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Determination of Glutamate and GABA Released by Mouse Embryonic Stem Cells Using HILIC-ESI-MS/MS

Author(s):

Haoyu Lv*, Yabin Tang, Fan Sun, Shimin An, Xinjie Yang, Wenbin Li, Xiaosheng Wang, Liang Zhu* and Hongzhuan Chen  

Abstract:


Background:It has been reported that embryonic stem cells (ESCs) expression functional receptors and biological elements synthesizing. However, specificity and high sensitivity detection means were required for confirming ESCs autocrine glutamate and γ-aminobutyric acid (GABA). Glutamate and GABA are water-souble and polar compounds which cannot be retained on a reversed phase C18 column, and their contents are often at a trace level. On the other hand, the biological matrix such as cell culture fluid contains a large number of amino acids, vitamins, carbohydrates, inorganic ions and other substances. Therfore, the main problem is selection of the chromatographic column and to avoid matrix interference.

Objective:To establish a rapid and reliable method for the simultaneous determination of glutamate and GABA released from embryonic stem cells based on analytical chemistry.

Methods: Glutamate and GABA released from mouse embryonic stem cells were determined on the basis of hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry ( HILIC- ESI- MS/MS ), using isotope internal standards and substitution matrix method.

Results: Undifferentiated embryonic stem cells autocrine glutamate and GABA and will reach releasing-reuptacking dynamic equilibriums at different time points. In contrast, neither glutamate nor GABA releasing could be detected from the MEFs, indicating the specificity release of the mESCs in the applied analytic method.

Conclusion: A novel quantitative method for the releasing of glutamate and GABA from mouse embryonic stem cells was developed. The method was validated simple, sensitive and selective.

Keywords:

glutamate, HILIC, LC-MS/MS, mouse embryonic stem cells, neurotransmitter, GABA

Affiliation:

Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, Department of basic medicine and clinical pharmacy, China Pharmaceutical University, 211198 Nanjing, Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai



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