GC-MS及GC测定茶叶中主要游离氨基酸的方法研究

doi:10.13305/j.cnki.jts.2010.06.007

Abstract

Abstract: 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.

Key words: GC-MS, GC-FID, MTBSTFA, theanine, free amino acid, tea

CLC Number:

TS272
O656.3

ZHANG Jia, WANG Chuan-pi, RUAN Jian-yun. Determination of Main Free Amino Acids in Tea by Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID)[J]. Journal of Tea Science, 2010, 30(6): 445-452.

References

[1] 倪君, 须海荣. 茶叶氨基酸检测方法的研究进展[J]. 茶叶, 2007, 33(2): 63-66.
[2] Sasaoka K, Kilo M.Studies on the biosynthesis of theanine in tea seedlings synthesis of theanine by the homogenate of tea seedlings[J]. Agric Biol Chem, 1963, 27(6): 467-468.
[3] Sasaoka K, Kilo M, Onishi Y.Some properties of the theanine synthesizing enzyme in tea seedlings[J]. Agric Biol Chem, 1965, 29(11): 984-988.
[4] Roach M C, Harmony M D.Determination of amino acids at subfemtomole levels by high-performance liquid chromatography with laser-induced fluorescence detection[J]. Anal Chem, 1987, 59(3): 411-415.
[5] 朱旗, 施兆鹏, 童京汉, 等. HPLC检测分析速溶绿茶游离氨基酸[J]. 茶叶科学, 2001, 21(2): 134-136.
[6] 李平, 宛晓春, 李健, 等. 茶氨酸的衍生化及毛细管电泳定量技术[J]. 茶叶科学, 2004, 24(2): 119-123.
[7] 张峻萍, 方从兵, 宛晓春, 等. 茶叶中茶氨酸的胶束电动毛细管电泳定量方法初步研究[J]. 茶叶通报, 2006, 28(3): 108-110.
[8] Blau Karl, Halket J M.Handbook of Derivatives for Chromatography (2nd Edn)[M]. John Wiley &Sons: Chichester, 1993: 11-99.
[9] Schwenk W F, Berg P J, Beaufrere B, et al. Use of t-butydimethylsilylation in the gas chromatographic/mass spectrometric analysis of physiological compounds found in plasma using electron-impact ionization[J]. Anal Biochem, 1984, 141(1): 101-109.
[10] Fortier G, Tenaschuk D, MacKenzie S L. Capillary gas chromatography micro-assay for pyroglutamic, glutamic and aspartic acids, and glutamine and asparagines[J]. J Chromatogr A, 1986, 361: 253-261.
[11] Shareef A, Angove M J, Wel J D.Optimization of silylation using N-methy-N-(trimethylsilyl)-trifluoroacetamide, N, O-bis-(trimethylsilyl)-trifluoro- acetamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide for the determination of the estrogens estrone and 17-ethinylestradiol by gas chromatography- mass spectrometry[J]. J Chromatogr A, 2006, 1108(1): 121-128.
[12] Schummer C, Delhommea O, Appenzellerb B M R, et al. Comparison of MTBSTFA and BSTFA in derivatization reactions of polar compounds prior to GC/MS analysis[J]. Talanta, 2009, 77(4): 1473-1482.
[13] Revelsky A I, Yashin Y S.Electron ionization and atmospheric pressure photochemical ionization in gas chromatography-mass spectrometry analysis of amino acids[J]. Mass Spectrom, 2003, 9(5): 497-507.
[14] Buch A, Glavin D P.A new extraction technique for in situ analyses of amino and carboxylic acids on Mars by gas chromatography mass spectrometry[J]. Planet Space Sci, 2006, 54(15): 1592-1599.
[15] Wood P L, Khan M A.Neurochemical analysis of amino acids, polyamines and carboxylic acids: GC-MS quantization of tBDMS derivatives using ammonia positive chemical ionization[J]. J Chromatogr B, 2006, 831(1/2): 313-319.
[16] Godber I M, Parsons R.Translocation of amino acids from stem nodules of Sesbania rostrata demonstrated by GC-MS in planta ¹⁵N isotope dilution[J]. Plant, Cell Environ, 1998, 21, 1089-1099.
[17] Persson J, Näsholm T.A GC-MS method for determination of amino acid uptake by plants[J]. Physiol Plant, 2001, 113(3): 352-358.
[18] 金海如. 丛枝菌根菌丝精氨酸双向运转并分解为鸟氨酸[J]. 中国科学, 2008, 38(11): 1048-1055.
[19] 何红波, 张威, 解宏图, 等. 测定土壤氨基糖和氨基酸手性异构体中氮同位素比值的气相色谱-质谱方法[J]. 土壤学报, 2009, 46(2): 290-298.
[20] Ruan J Y, Gerendás J, Härdter R, et al. Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea (Camellia sinensis)[J]. plants Ann Bot, 2007, 99(2): 301-310.
[21] Deng W W, Ogita S, Ashihara H.Biosynthesis of theanine (γ-ethylamino-glutamic acid) in seedlings of Camellia sinensis[J]. Phytochem, 2008, 1(2): 115-119.
[22] Oh K, Kato T, Xu H L.Transport of nitrogen assimilation in xylem vessels of green tea plants fed with NH₄-N and NO₃-N[J]. Pedosphere, 2008(18): 222-226.
[23] Ruan JY, Härdter R, Gerendás J.Impact of nitrogen supply on carbon/nitrogen allocation: a case study on amino acids and catechins in green tea (Camellia sinensis (L.) O. Kuntze)[J]. Plant Biology, 2010, 12(5): 724-734.

Determination of Main Free Amino Acids in Tea by Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID)

PDF (PC)

Knowledge

Cited

Abstract

Cite this article

share this article

References

Related Articles 15

Metrics

Comments

Recommended 10

[1]	GAO Jianjian, CHEN Dan, PENG Jiakun, WU Wenliang, CAI Liangsui, CAI Yawei, TIAN Jun, WAN Yunlong, SUN Weijiang, HUANG Yan, WANG Zhe, LIN Zhi, DAI Weidong. Comparison on Chemical Components of Yunnan and Fuding White Tea Based on Metabolomics Approach [J]. Journal of Tea Science, 2022, 42(5): 623-637.
[2]	CHEN Qiyu, MA Jianqiang, CHEN Jiedan, CHEN Liang. Genetic Diversity of Mature Leaves of Tea Germplasms Based on Image Features [J]. Journal of Tea Science, 2022, 42(5): 649-660.
[3]	LI Yanchun, WANG Hang, LI Zhaowei, YE Jing, WANG Yixiang. Ameliorative Effect of Several Measures on Soil Physicochemical Properties and Microbial Community Structures in Acidified Tea Gardens [J]. Journal of Tea Science, 2022, 42(5): 661-671.
[4]	SUN Yue, WU Jun, WEI Chaoling, LIU Mengyue, GAO Chenxi, ZHANG Lingzhi, CAO Shixian, YU Shuntian, JIN Shan, SUN Weijiang. Screening of Tea Germplasm Resistant to Matsumurasca onukii and Dendrothrips minowai Priesner and Analysis of Resistance-related Factors [J]. Journal of Tea Science, 2022, 42(5): 689-704.
[5]	CHEN Yuhong, GAO Ying, HAN Zhen, YIN Junfeng. Analysis of the Saponin Contents and Composition in Tea Seeds of Different Germplasms [J]. Journal of Tea Science, 2022, 42(5): 705-716.
[6]	CHEN Hui, YANG Liling, CHEN Jinhua, HUANG Jian'an, GONG Yushun, LI Shi. Effect of Temperature-controlled Pile-fermentation on Aroma Quality of Primary Dark Tea [J]. Journal of Tea Science, 2022, 42(5): 717-730.
[7]	LI Zheng, LIU Ding, HUO Zenghui, CHEN Fuqiao. Analysis on the Competitiveness and Complementarity of Tea Trade between China and RCEP Members [J]. Journal of Tea Science, 2022, 42(5): 740-752.
[8]	WANG Yuyuan, LIU Renjian, LIU Shaoqun, SHU Canwei, SUN Binmei, ZHENG Peng. Expression Analysis and Functional Identification of CsTT2 R2R3-MYB Transcription Factor in Tea Plants [J]. Journal of Tea Science, 2022, 42(4): 463-476.
[9]	LI Jing, LIN Cairong, HUANG Yan, DENG Xuming, WANG Yiqing, SUN Weijang. Effects of Tea Polyphenols on Agrobacterium-mediated Plant Genetic Transformation System [J]. Journal of Tea Science, 2022, 42(4): 477-490.
[10]	ZHAO Dongwei. Nomenclature, Typification, and Natural Distribution of Camellia sinensis var. assamica (Theaceae) [J]. Journal of Tea Science, 2022, 42(4): 491-499.
[11]	LIU Jianjun, ZHANG Jinyu, PENG Ye, LIU Xiaobo, YANG Yun, HUANG Tao, WEN Beibei, LI Meifeng. Effects of Light Waves on the Aroma Substances of Fresh Tea Leaves in Summer and Autumn During Spreading and the Quality of Final Green Tea [J]. Journal of Tea Science, 2022, 42(4): 500-514.
[12]	ZHANG Yinggen, XIANG Lihui, CHEN Lin, LIN Qingxia, SONG Zhenshuo, WANG Lili. Effects of Air Conditions Preset for Withering on Flavor Quality and Chemical Profiles of White Teas [J]. Journal of Tea Science, 2022, 42(4): 525-536.
[13]	LI Ziqiang, YANG Mei, ZHANG Xinzhong, LUO Fengjian, ZHOU Li, LOU Zhengyun, SUN Hezhi, WANG Xinru, CHEN Zongmao. Residue Determination of Sixteen Pesticides in Green Tea by UPLC-MS/MS [J]. Journal of Tea Science, 2022, 42(4): 537-548.
[14]	FANG Mengrui, LÜ Jun, RUAN Jianyun, BIAN Lei, WU Chuanyu, YAO Qing. Tea Buds Detection Model Using Improved YOLOv4-tiny [J]. Journal of Tea Science, 2022, 42(4): 549-560.
[15]	SUN Ying, CHEN Xin, YANG Hua, YING Jian, SHAO Danqing, LÜ Xiaohua, XIAO Jie, CHEN Zhixiong, LI Song, QIN Junjie, ZHENG Bin, GAO Jianshe. Clinical Trial on the Effect of Drinking Jinhua Xiangyuan Tea for 3 Months on the Improvement of Glucose and Lipid Metabolism in A Small Sample Hyperlipidemia Population [J]. Journal of Tea Science, 2022, 42(4): 561-576.