Establishment And Application Of Two-dimensional Liquid Chromatography For Five Endogenous Amino Acid Neurotransmitters

Amino acid neurotransmitters(AANTs)interact with biological amines,peptides and other neurotransmitters to maintain the dynamic balance of excitability in animals through a series of processes of synthesis,transformation and disappearance.In view of the fact that the changes in the content of these neurotransmitters in the body will affect their relative balance in the biological matrix,which in turn determines the excitability level of neurons,the determination of the changes in the content of neurotransmitters is closely related to the physiological function and pathological changes of the brain.Therefore,it is necessary to establish a neurotransmitter detection method to analyze the content of biological samples.At present,endogenous AANTs mainly include aspartic acid(Asp),glutamic acid(Glu),glycine(Gly),taurine(Tau)and gamma-aminobutyric acid(GABA),and most of them are analyzed by high performance liquid chromatography(HPLC)coupled with fluorescence,mass spectrometry and ultraviolet detection systems,followed by capillary electrophoresis(CE)and gas chromatography(GC).Therefore,in this paper,a two-dimensional liquid chromatography(2D-LC)method was developed for the simultaneous detection of five endogenous AANTs,which provides a basis for clinical application and determination of amino acids(AA)in various biological samples.A method for the simultaneous determination of Asp,Glu,Gly,Tau and GABA in animal blood and brain by two-dimensional liquid chromatography(2D-LC)combined with ultraviolet detection was established for the first time.After the sample was collected,it was precipitated and centrifuged,and then 4-fluoro-7-nitrobenzofurazan(NBD-F)was used to label AANTs on the corresponding fluorescent derivatives.A derivatization system of 100μL AANTs/sample supernatant,350 μL borate buffer(10 mmol/L)and 50 μL NBD-F(10mmol/L)working solution was designed for derivatization reaction.The reaction products were injected into the extraction column(one-dimensional column: SNCB 50 mm*4.6mm*5 μm column)for primary separation,partial impurity removal,target enrichment and automatic transfer into the analytical column(two-dimensional chromatographic column:SBR3 200 mm*4.6 mm*5 μm column),so as to realize the on-line extraction and complete separation of target components.The aqueous phase consisted of 0.02 mol/L sodium dihydrogen phosphate and disodium hydrogen phosphate buffer(p H = 6.8～6.9),and the organic phase consisted of methanol-acetonitrile(3:1,V/V),were used to flow through the analytical column.The mobile phase flowing through the extraction column was methanol and 0.02 mol / L sodium dihydrogen phosphate and sodium dihydrogen phosphate buffer solution(p H = 6.8 ～ 6.9)(15:85,V/V).After the methodology verification and blank content determination under the above conditions,the acute toxicity test was designed by intragastric administration,and the rats were given single administration,and the drug effects of gelsenicine on AANTS were studied by 2D-LC-UV method.The results showed that the method showed good selectivity,and the correlation coefficient of analyte calibration curve was more than 0.99.This method exhibited good selectivity,and the correlation coefficients for the analyte calibration curves of were > 0.99.The intra-and inter-day precisions were ≤16.03,and the accuracies were in the range of 70.59%-116.20%.The system realizes the rapid detection and stability quantification of the five AANTs,which proves that the alternative dilution method is feasible.In addition,the established method has been successfully applied to the analysis of rat serum and pig plasma,whole brain and brain regions(hippocampus,cortex,striatum,cerebellum,brain stem and hypothalamus)of rats and pigs.The results showed that there were some differences between the brain regions of rats and pigs.The content of AANTs by gelsenicine was related to the difference between male and female and the location of distribution,and pointing to the spinal cord may be related to the toxic mechanism of gelsenicine.The system has high loading capacity,excellent resolution,and good peak shape and is not affected by other endogenous substances.This method is expected to provide applicability for the determination of AANTs in pharmacology,pharmacy and clinical research of neuroscience,and also provide methodological research for the application of Gelsemium in the study of neurodrugs.