研究了N-（特丁基二甲基硅烷）-N-甲基三氟乙酰胺（≥95%;MTBSTFA）含叔丁基二甲基氯硅烷（1%;TBDMSCl）衍生、气相色谱（GC）和气相质谱联用仪（GC-MS）测定茶叶中游离氨基酸的方法,比较了衍生温度、时间、酸度及辅助试剂等对衍生效率的影响。以正缬氨酸为内标,乙腈为辅助衍生试剂,衍生温度110℃,衍生时间30 min,可以定量检测茶叶中主要的游离氨基酸。茶氨酸产生3个衍生峰,经质谱鉴定第1个峰（按出峰时间）为环状衍生峰,第2个峰为一个N端没有衍生,第3个峰为完全衍生峰。各氨基酸在10~1 000 nmol范围内线性良好（相关系数均大于0.99）,对茶叶样品测定结果与氨基酸自动分析仪基本一致,添加回收试验表明,茶氨酸、谷氨酸、谷氨酰胺、丝氨酸、天冬氨酸等的回收率为85%~108%。结果表明,试验建立的GC-MS及GC-FID方法操作简便、准确,重现性好,适用于茶叶中主要游离氨基酸的分离检测,同时GC-MS方法还可用于15N标记的氨基酸的检测,为吸收代谢实验提供研究基础。
Free amino acids are important metabolites and contributors in green tea quality. Methods were developed to quantitatively analyze free amino acids in particular theanine in tea by Gas Chromatography/Mass Spectrometry (GC-MS) and Gas Chromatography/Flame Ionization Detector (GC-FID). The derivative reagent used in this study was N-tertbutyldimethylsily-N-methytrifluroacetamide (MTBSTFA, ≥95%) with tert- butyldimethylsilyl chloride (TBDMSCl, 1%). Conditions for derivatization (reagent, temperature, time, and acidity) were optimized to give high efficiency. The optimized procedure was as follows: dried amino acids standards or tea extract (water) under N2 gas were re-dissolved 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) within the ranges of 10 to 1 000 nmol. Three peaks were detected for theanine. The MS data indicated that only the third peak (according to peak times) was the completely derivatized product while the first was cyclic and the second was a product of one N-end non-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% ranges for spiked tea extract. The developed GC-MS method provided a potential methodology to investigate nitrogen and theanine metabolism in tea plants by quantifying the abundance of 15N labeled amino acids.
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