The Study On The Quantitative Methodology For Glutamate And ?-Aminobutyric Acid Released From Embryonic Stem Cells Using HILIC-MS/MS

It has been reported that embryonic stem cells(ESCs)expression functional receptors and biological elements synthesizing and releasing glutamate and ?-aminobutyric acid(GABA).However,specificity and high sensitivity detection means were required for confirming wether or not ESCs autocrine glutamate and GABA.At present,the commonly used detection methods are chromatography tandem ultraviolet visible / fluorescence detection method,etc..These methods might introduce impurities and reduce detection sensitivity due to the need of derivatization treatment or addition of ion pair reagents,which can affect the reliability of the results.By contrast,liquid chromatography-mass spectrometry has gradually become one of the main methods for the analysis of amino acids.Because glutamate and GABA are polar compounds,which cannot be retained in a reversed phase C18 column,a novel analytical method was developed on the basis of hydrophilic interaction chromatography(HILIC),using isotope internal standards and substitution matrix method.This method significantly improved the retention time of polar and ionic compounds,got rid of the influence of ion suppression,and increased the ionization efficiency of mass spectrometry.At the same time,HILIC was effective to solve the problem of weak retention of polar substances in the traditional C18 chromatographic column,and on the other hand,the lower viscosity of the mobile phase made the separation speed faster than the reversed phase chromatography.The method was validated simple,sensitive and selective.It was found that ESCs autocrine glutamate and GABA until a releasing-reuptaking dynamic equilibrium is reached.It will provide methodology means and data trail for the biological research of stem cells.