Study On The Method For Measuring The Main Free Amino Acids And Their ¹⁵N Abundance In Tea By Gas Chromatography- Mass Spectrometry (gc-ms)

Free amino acids are important metabolites and contributor to tea quality. L-Theanine is a particular non-protein amion acid in tea,in addition to this, the main free amino acids in tea include Glu, Gln, Ala, Ser, Asp and so on. And amin acids have close relation to flavor of tea, the content of amino acids influence the quality of tea directly. As the methods are improved rapidly in recent years, and currently we primarily use instrument for the determination and identification of amino acids. But there is no report that use Gas Chromatography-Mass Spectrometry(GC-MS) determination of main free amino acids in tea. The dissertation consists of two parts : Part one is research and established the mothed about Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID) in determination of main free amino acids in tea. The derivative reagent used in this study was N-tertbutyldimethylsily N-methytrifluroacetemide (MTBSTFA,≥95%) with tert-butyldimethylsilyl chloride (TBDMSCl, 1%). Conditions of reagent, temperature, time, and acidity for derivatization were optimized to give high efficiency. The optimized procedure was as follows: dried amino acids standards or tea extract (water) under N2 gas were redissolved in acetonitrile (ACN) and derivatized with MTBSTFA (1:1 to ACN) at 110℃for 30 min. Analysis of pure amino acid standards showed good linearity (r2≥0.99) from 10 to 1000 nmol. Three peaks were detected for theanine with GC and MS data indicated that only the third peak was the completely derivatized product while the second peak was a product of one N-end derivatization. Free amino acids in tea sample were quantified by the developed methods and the results were comparable to those analyzed by Amino Acids Autoanalyzer. Recoveries of theanine, alanine, serine, aspartate, glutamate, and glutamine were 85%-108% range for spiked tea extract.Part two is about use Gas Chromatography-Mass Spectrometry (GC-MS) analysis the main free amino acids in tea (Thea, Ala, Glu, Gln )which absorbed different nitrogen source labelled stable isotope 15N. The application of stable isotopic tracer technology is universally, but the study of isotopomer distributions in tea has not been reported. GC-MS is a rapid method that provides rich information on isotopomer on isotopomer distributions for metabolic flux analysis.After derivatized with MTBSTFA , analysed the characteristic ion peak M-57, mass spectroscopy gave rise to characteristic spectra for all analysed compounds and established the detection method preliminary. Used Amino acid standards enriched with different proportions (0, 20, 40, 60, 80 and 100%) of universally (U) 15N-labelled Ala, Glu, Gln.(amion acids containing > 98% 15N at all nitrogen positions), were used to calibrate the method for isotopic enrichment of the individual amino acids. The tea plant were placed in a greenhouse ,and were supplied with 15NH4+ for 48h. A comparative analysis between control and 48h show that there are difference in labelled amino acids (theanine not very obviously). The developed GC-MS method provided a potential methodology to investigate nitrogen metabolism in tea plants by quantifying the abundance of 15N labeled amino acids.