Journal of Food Composition and Analysis

Transcription

1 Journal of Food Composition and Analysis 22 (2009) Contents lists available at ScienceDirect Journal of Food Composition and Analysis journal homepage: Original Article A novel colorimetric determination of free amino acids content in tea infusions with 2,4-dinitrofluorobenzene Lin Chen a,b, Qi Chen a, Zhengzhu Zhang a, Xiaochun Wan a, * a Key Laboratory of Tea Biochemistry & Biotechnology, Ministry of Agriculture & Ministry of Education, Anhui Agricultural University, West 130 Changjiang Road, Hefei, Anhui , China b Tea research institute, Fujian Academy of Agricultural Sciences, Fu an, Fujian , China ARTICLE INFO ABSTRACT Article history: Received 6 December 2007 Received in revised form 23 July 2008 Accepted 2 August 2008 Keywords: Tea infusions Amino acids Colorimetric method 2,4-dinitrofluorobenzene Ninhydrin Food composition A simple procedure is presented for the estimation of free amino acids content in tea infusions based on the reaction of amino groups with 2,4-dinitrofluorobenzene (DNFB) and subsequent measurement of the dinitrophenyl (DNP)-amino acids extracted from reaction mixtures into ethyl acetate at 420 nm. The contents of free amino acids in various tea infusions determined by the proposed method are almost consistent with an existing ninhydrin colorimetric method, but the derivatization reaction with DNFB is more sensitive, and the spectral curves of every resulting product will be completely overlapped in the range of nm during a week of storage. The absorbance values of DNP-derivatives (Y) are linear with the concentrations of amino acids (X) in the range of mg/ml, and their linear regression equations are Y = X (R 2 = ) for L-theanine and Y = X (R 2 = ) for L-glutamic acid, respectively. A complete reaction system contains 1.0 ml of 0.2 mol/l sodium bicarbonate solution, 1.0 ml of 1% DNFB (a 1.0-mL portion is dissolved in 100 ml of 1,4-dioxane) and 1.0 ml of each sample, incubated at 60 8C for 40 min in the dark. Under the described standard conditions, the average recoveries of amino acids in infusions of black tea, green tea, Oolong tea, yellow tea, white tea and dark tea are % with R.S.D. of % estimated by L-theanine and % with R.S.D. of % estimated by L-glutamic acid. ß 2009 Published by Elsevier Inc. 1. Introduction Free amino acids are regarded as important taste components in terms of tea quality, especially for green tea (Horie and Kohata, 2000). Although 26 varieties of amino acids could be found and identified in tea plants (Wang, 1984), most of them in tea leaves were theanine, glutamic acid, aspartic acid, serine, glutamine, alanine, arginine, threonine (Takeo, 1980), and theanine accounted for about 60 70% of total contents of free amino acids in spring shoots (Takeo, 1981). Theanine is primarily responsible for umami (a brothy or savory) taste of green tea, similar to that of sodium glutamate (Juneja et al., 1999). Newly interests have been peaked at its positive effects on immune system responsiveness to infection, blood-pressure reduction, relaxation, neuroprotection, and modulation of chemotherapy (Eschenauer and Sweet, 2006). As a unique amino acid identified only in the tea plants (Sakato, 1950), with the exception of a kind of mushroom, Xerocomus badius (Casimir et al., 1960), and certain species of genus Camellia, C. japonica and C. * Corresponding author. Tel.: ; fax: address: tealab@ahau.edu.cn (X. Wan). sasanqua (Tsushida and Takeo, 1984), theanine also demonstrated the same chemical characters with most amino acids, such as color reaction with ninhydrin (Li and Hu, 1989; Nakagawa and Anan, 1979; Selvendran and Selvendran, 1973), derivatization by 5- dimethylamino-1-naphthalenesulfonyl chloride (dansyl-cl) (Otsuki et al., 1979), o-phthalaldehyde (OPA) (Goto et al., 1993; Tsushida and Takeo, 1983; Ying et al., 2005), 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (Zhu et al., 2001), 9-fluoromethoxycarbonylglycine chloride (FMOC-Cl) (Desai and Armstrong, 2004), phenylisothiocyanate (PITC) (Thippeswamy et al., 2006), 2,4- dinitrofluorobenzene (DNFB) (Friedman et al., 2007), etc. These properties have been applied to analyze the compositions of free amino acids and theanine content in tea products, often combined with isolation and quantification by paper chromatography, thinlayer chromatography, automatic amino acid analyzer, highperformance liquid chromatography (HPLC) or capillary electrophoresis. However, when it came to measure total content of free amino acids in tea extracts, the ninhydrin colorimetric method was preferably adopted just as for determination of compounds containing amino groups in food and pharmaceutical products (Sun et al., 2006). Teas are usually categorized into six types, black tea, green tea, Oolong tea, yellow tea, white tea and dark tea in China /$ see front matter ß 2009 Published by Elsevier Inc. doi: /j.jfca

2 138 L. Chen et al. / Journal of Food Composition and Analysis 22 (2009) Because of predominant content of L-theanine in various tea infusions, L-theanine and its precursor, L-glutamic acid were commonly used to prepare standard curves to estimate total free amino acids content with this colorimetric determination (Liang et al., 2003; Sasaoka et al., 1963). Since the description by Sanger of the easy reactivity of 2,4- dinitrofluorobenzene with free amino groups (Sanger, 1945), many applications of this reagent have been reported. It has become a routine tool in the analysis of the amino end-groups of proteins, and of amino acids in protein hydrolysates (Dubin, 1960). Because the fluorine and nitro groups withdraw electron density from the benzene ring, a carbon atom in the ring undergoes nucleophilic attack by the N-terminal nitrogen of an amine group, forming compounds (dinitrophenyl (DNP)-derivatives) that will strongly absorb light in the UV and/or visible spectrum. As a commercial product, the appearance of DNFB is colorless liquid or light yellow crystal. Absorption spectrum of DNFB will change in solutions with different ph conditions, but not to its derivatives of amino acids, and these phenomena have not been reported yet. Moreover, DNPamino acids can be completely extracted into ethyl acetate from acidified mixtures of water and 1,4-dioxane. Thus, compared with the existing colorimetric determination based on ninhydrin reaction, a novel analytical procedure was developed to estimate free amino acids content in tea infusions after derivatization with DNFB in this paper. 2. Materials and methods 2.1. Samples and chemicals Black tea (Qimen Gongfu), green tea (Yuexi Cuilan), Oolong tea (Fujian Tieguanyin), yellow tea (Huoshan Huangdacha), white tea (King of Mudan) and dark tea (Guangxi Liubaocha) were collected from their original regions. L-Theanine with purity of 99.3% was obtained from Jintan Qianyao Pharmaceutical Material Factory (Jiangsu province, China). L-Glutamic acid was a biochemical reagent with purity of 98.5%. DNFB was a product of Tokyo Chemical Industry Co. (Japan). Trifluoroacetic and acetonitrile were of HPLC grade purchased from Tedia Company (USA). Ninhydrin and other chemicals used in this experiment were of analytical grade obtained in China. Unless otherwise specified, all reagents were prepared with demineralized water purified by Pall PL5243 PURELAB Classic water purification system (USA) Preparation of tea infusions All tea samples were ground into minute particles by an analytical mill (IKA All Basic, German). Each of them (0.300 g) was placed into a 50-mL tube to which added with 40 ml of water. The tubes covered with stoppers were heated on a water bath (100 8C) for 60 min, and the cooled samples were adjusted to 40 ml with water. Filtered through two layers of Double-ring no.102 filter paper (Xinhua Paper Industry Co. Ltd., Hangzhou, China) by reduced pressure, these tea infusions were used for measuring the contents of free amino acids and L-theanine Determination of free amino acids by DNFB derivatization In a 7-mL plastic centrifugation tube, 1.0 ml of each tea infusions was added (to avoid disturbance from components soluble in ethyl acetate, all tea infusions were pre-extracted with five volumes of ethyl acetate before analysis.). This was followed by addition of 1.0 ml of 0.2 mol/l sodium bicarbonate solution and 1.0 ml of 1% DNFB (a 1.0 ml portion was dissolved in 100 ml of 1,4-dioxane). The probes in the sealed tubes were then placed on a water bath (60 8C) and left to stand for 40 min in the dark. All derivative reactions were stopped by addition of 0.5 ml of 1 mol/l HCl. The resulting DNP derivatives (0.75 ml) were extracted with 5 ml ethyl acetate. After they were stand for 10 min, the UV spectrum characters of upper phases were analyzed with ethyl acetate as reference (HITACHI U-3010 Spectrophotometer coupled with UV Solution 2.0 workstation, 10 mm light path length.) Determination of free amino acids by ninhydrin reaction In a 25-mL volumetric flask, 1.0 ml of each tea infusions was added. This was followed by addition of 0.5 ml of 1/15 mol/l phosphate buffer solution (ph 8.04) and 0.5 ml of 2% ninhydrin solution containing 0.8 mg/ml of SnCl 2 2H 2 O. The mixtures in the volumetric flasks were then placed on a boiling water bath for 15 min. The probes were quickly cooled with cold water, and adjusted to 25 ml with water. After they were left to stand still for 10 min, the absorbance values of these blue-purple products were measured against a reagent blank (tea infusions replaced by the equal volume of water was carried through the procedure). The other details referred to GB/T , a national standard method used to determine free amino acids content of tea infusions in China Determination of L-theanine content by HPLC Contents of L-theanine in the various tea infusions were determined with SHIMADZU Prominence HPLC consisting of LC- 20AD pump, DGU-20A5 degasser, and SPD-M20A diode array detector (200 nm), operated with LC solution workstation. The chromatographic analysis was performed on a C 18 reverse phase column (Spherigel, 250 mm 4.6 mm id, particle size 5 mm) at 25 8C. The mobile phase consisted of 0.05% trifluoroacetic acid water (v/v) (A) and 50% acetonitrile water (v/v) (B). 5 ml of each tea infusions was manually injected, followed by filtration through a 0.45-mm filter. Solvent A had been maintained at flow rate of 1.0 ml/min for 15 min, then the column was eluted with solvent B for 20 min before it was equilibrated 10 min for another analysis. The total transition time between A and B was 10 min. 3. Results and discussion 3.1. Spectral characters of DNFB and DNP-amino acids The color of DNFB appears yellow-green in alkaline solution, and colorless in neutral or acidic solution. These phenomena are consistent with its changes of spectral absorbance in solutions with different ph conditions (Fig. 1). Yellow-green compounds could be formed when DNFB was reacted with L-theanine and L- glutamic acid, and the color of DNP derivatives would not fade in neutral or acidic solution. To get rid of disturbance of DNFB for analysis of amines, excess DNFB was recommended to be hydrolyzed by strong alkali, and that the dinitrophenylamine could be extracted from the alkaline aqueous reaction mixture into an immiscible solvent such as cyclohexane for spectrophotometric determination (Kolbezen et al., 1962). Because of the existence of 1,4-dioxane, which could be well mixed with DNFB and water, this procedure was not suitable for our experiment to extract DNP-amino acids into cyclohexane. On the contrary, 0.5 ml of 1 mol/l HCl was added into a reaction mixture, thereafter DNFB and the derivatives of amino acids could be both extracted into ethyl acetate. Because the mixture became an acidified solution after addition of HCl, no background

3 L. Chen et al. / Journal of Food Composition and Analysis 22 (2009) Table 1 Orthogonal experimental scheme for derivatization reaction and the absorbance of resulting DNP-amino acids. Treatment NaHCO 3 (mol/l) Temperature (8C) Time (min) Absorbance of DNP-amino acids (l = 420 nm) Fig. 1. Spectral absorbance curves of DNFB. (a) 1.0 ml 0.2 mol/l NaHCO ml 1% DNFB ml H 2 O; (b) 1.9 ml H 2 O ml 1% DNFB; (c) 1.0 ml 0.2 mol/l HCl ml 1% DNFB ml H 2 O and (d) 1.0 ml 0.2 mol/l HCl ml 1,4- dioxane ml H 2 O (reference) Not detected a Not detected b Not detected a Not detected b Not detected a Not detected b Not detected a Not detected b a b a b a b a b a b a b a b a b a b a b a b a b a DNP-theanine. b DNP-glutamic acid. Fig. 2. Spectral absorbance curves of DNP-amino acids. (a) DNFB & derivatives of L- theanine extracted by ethyl acetate; (b) DNFB & derivatives of L-glutamic acid extracted by ethyl acetate; (c) DNFB extracted by ethyl acetate and (d) ethyl acetate (reference). effects from DNFB existed in the extractions of ethyl acetate when the absorbance values of DNP-theanine and DNP-glutamic acid were measured over 420 nm (Fig. 2) Optimization of conditions for derivatization reaction Both amino acids and amines could be derivatized by DNFB in weak basic solutions, but the need for time/temperature control would vary according to the characters of compounds. In a typical experiment reported by Lockhart to derivatize apliphatic amines, DNFB (0.15 ml) in 0.2 ml of ethanol, equal volume of 0.2 mol/l sodium bicarbonate solution was mixed with 0.1 ml amines containing 10 mg of the amine or its hydrochloride. After standing for 15 min at room temperature, the solution was placed at 105 8C in a sealed tube for 2 h (Lockhart, 1956). Moreover, procedures presented by Friedman et al. to analyze theanine content of tea leaves, 0.1 ml of DNFB and 2 ml of 1% sodium bicarbonate solution were added to the residues containing theanine, and the mixtures were allowed to stand in the dark for 3 h at 40 8C (Friedman et al., 2007). These time-consuming methods with no definite temperature control made us design an orthogonal experimental scheme with three factors and four levels to investigate the effects of concentrations of sodium bicarbonate solution, temperature and time for reactions on the absorbance values of DNP-amino acids in extractions of ethyl acetate (Table 1). A variance analysis was carried out and its result showed that concentration of sodium bicarbonate was of notable effects on absorbance values of DNP-amino acids in ethyl acetate at 420 nm (P < 0.01), but not the other two factors. It could be seen that DNPamino acids in extractions of ethyl acetate would not be detected from treatment 1 to treatment 4 when equal volume of water was added into the reaction system instead of sodium bicarbonate solution. In addition, the absorbance values of extractions tended to be increased by adding higher concentrations of sodium bicarbonate solutions. Although the average absorbance values of DNP-theanine (0.997 mg/ml) and DNP-glutamic acid (0.937 mg/ ml) were obtained with 1 ml of 0.4 mol/l sodium bicarbonate solution added into reaction systems, background effects from DNFB in ethyl acetate could be also found with the absorbance values less than at 420 nm. Therefore, the 11th treatment would be the optimum conditions for derivatization reaction, by which the highest absorbance values of DNP-theanine (0.999 mg/ ml) and DNP-glutamic acid (0.929 mg/ml) could be measured with no interference from DNFB. Given the possible effects from reaction time on the derivatization of the other amino acids in tea infusions, the treatment by incubating reaction systems at 60 8C for 40 min with addition of 1 ml of 0.2 mol/l sodium bicarbonate solution described in the previous method was applied in the latter analyses Comparison of total free amino acids content estimated by DNFB method and ninhydrin method L-theanine and L-glutamic acid standard solutions were prepared over seven different concentrations, which were 0.02 mg/ml, 0.10 mg/ml, 0.20 mg/ml, 0.40 mg/ml, 0.60 mg/ml, 0.80 mg/ml and 1.00 mg/ml. The studies indicated that gradient changes of absorbance values of DNP-amino acids could be found between 420 nm and 480 nm, and the absorbance values of DNPderivatives were linear with the concentrations of amino acids in the range of mg/ml at 420 nm (Figs. 3 and 4). With these different concentrations of amino acids, the standard calibration curves for ninhydrin method and HPLC (Ltheanine) were also established according to corresponding manipulations described in Sections 2.4 and 2.5. HPLC analysis was of the same linear range with the peak area (Y 0 ) against the concentration of L-theanine (X 0 ) at 200 nm as derivative reaction, but the color reaction with ninhydrin was in the range of 0.10

4 140 L. Chen et al. / Journal of Food Composition and Analysis 22 (2009) Fig. 3. Spectral absorbance curves of derivatives with different concentrations of L- theanine (mg/ml). Fig. 6. Spectrum of compounds formed by ninhydrin reaction with different concentrations of L-glutamic acid (mg/ml). Fig. 4. Spectral absorbance curves of derivatives with different concentrations of L- glutamic acid (mg/ml) mg/ml at 567 nm (maximum absorbance values could be reached at this wavelength) (Figs. 5 and 6). The estimated contents of free amino acids and contents of L- theanine in various tea infusions were listed in Table 2. It was shown that no significant differences between two methods to predict the free amino acids content with L-theanine or L- glutamic acid, except that some higher content could be found in black tea, and free amino acids could be also detected in dark tea with DNFB method. Although the correlation coefficient Fig. 5. Spectrum of compounds formed by ninhydrin reaction with different concentrations of L-theanine (mg/ml). between L-theanine content in each tea infusions determined by HPLC and the free amino acids content estimated with L-theanine reached by DNFB method and by ninhydrin method, the mean contents of L-theanine only accounted for mg/ml. Therefore, it could be concluded that most of yellow-green derivatives (DNFB method) or blue-purple compounds (ninhydrin method) were produced from the other amino acids. Theoretically, other compounds with amine groups in tea infusions would contribute to higher estimation of free amino acids content by ninhydrin reaction, but it would be less interference for DNFB method, because DNP-amino acids were determined by extraction into ethyl acetate, and derivatives of short peptides, soluble proteins and their complexes were left in the lower phase with 1,4-dioxane. According to these similarities and differences between two methods, it would be more sensitive and accurate for estimation of free amino acids content in tea infusions by the derivative method Validation of DNFB method A series of sample analyses were performed to validate the performance of the DNFB method. Precision was evaluated from replicate determinations (n = 5) performed on the same day for each tea infusions. The mean contents of free amino acids in various tea infusions were shown in Table 3. The known quantities of L-theanine or L-glutamic acid were added to various tea infusions, and the recoveries were also calculated by comparing the found amount of standards to those of added. The results indicated that relative standard deviation (R.S.D.) of free amino acids content in various tea infusions were from 0.507% to 1.299% estimated by L-theanine and from 0.500% to 1.271% estimated by L- glutamic acid. Average recoveries of tea infusions added with standards were from % to % for L-theanine and from % to % for L-glutamic acid. As a routine method to analyze free amino acids content in tea infusions, reproducible results would be obtained by the described ninhydrin method, but the colorimetric determination should be finished not more than 2 h because of the instability of blue-purple products. However, a continuous study indicated that the spectral curves of DNP-amino acids (standards or free amino acids in various tea infusions) extracted into ethyl acetate stored at dark place with room temperature could be completely overlapped in the range of nm with their corresponding primitive curves in a week. Therefore, free amino acids content in large numbers of tea samples would rather be determined by DNFB method for its good reproducibility and stability.

5 L. Chen et al. / Journal of Food Composition and Analysis 22 (2009) Table 2 Content of free amino acids and L-theanine in various tea infusions (mg/ml). Analyte Black tea Green tea Oolong tea Yellow tea White tea Dark tea The linear regression equation Free amino acids a a a a a a Y = X (R 2 = ) a (DNFB method) b b b b b b Y = X (R 2 = ) b Free amino acids a a a a a Not detected a,b Y = X (R 2 = ) a (Ninhydrin method) b b b b b Y = X (R 2 = ) b L-theanine Y 0 =6E + 06X (R 2 = 0.999) a Contents of free amino acids estimated by L-theanine. b Contents of free amino acids estimated by L-glutamic acid. Table 3 Precision and recovery of experiments. Sample 4. Conclusions The contents of free amino acids in tea infusions could be determined by conventional color reaction with ninhydrin, but this method was less sensitive and more unstable than the DNFB method presented in this paper, which was conducted under a convenient temperature (60 8C). Because the color of DNP-amino acids remained and did not change to DNFB in acidified solutions, the contents of free amino acids in tea infusions could be well determined and estimated by L-theanine or L-glutamic acid, even without essentially complete removal of excess DNFB. Acknowledgments The research work was supported by a grant of the National Science Foundation of China (No ) and in part by the Major State Basic Research Development Program of China (No. 2004CCA06400). The authors would like to thank Zai-Xin Hua, an associate professor in our lab of tea quality, for her providing various tea samples, and Jiang-Yu FANG, a postdoctoral fellow in Anhui Agricultural University, for helpful advices on this manuscript. References Average (mg/ml) R.S.D. (%) Average recovery (%) R.S.D. of recovery (%) Black tea a a a a b b b b Green tea a a a a b b b b Oolong tea a a a a b b b b Yellow tea a a a a b b b b White tea a a a a b b b b Dark tea a a a a b b b b a Contents of free amino acids estimated by L-theanine. b Contents of free amino acids estimated by L-glutamic acid. Casimir, J., Jadot, J., & Renard, M. (1960). Separation and characterization of N-ethylgamma-glutamine in Xerocomus badius (Boletus ladius). Biochimica et Biophysica Acta, 39, Desai, M. J., & Armstrong, D. W. (2004). Analysis of derivatized and underivatized theanine enantiomers by high-performance liquid chromatography/atmospheric pressure ionization-mass spectrometry. Rapid Communications in Mass Spectrometry, 18, Dubin, D. T. (1960). The assay and characterization of amines by means of 2,4- dinitrofluorobenzene. The Journal of Biological Chemistry, 235, Eschenauer, G., & Sweet, B. V. (2006). Pharmacology and therapeutic uses of theanine. American Journal of Health-System Pharmacy, 63(26), Friedman, M., Mackey Bruce, E., Kim, H. J., Lee, I. S., Lee, K. R., Lee, S. U., Kozukue, E., & Kozukue, N. (2007). Structure-activity relationships of tea compounds against human cancer cells. Journal of Agricultural and Food Chemistry, 55, Goto, T., Horie, H., & Mukai, T. (1993). Analysis of major amino acids in green tea by high performance liquid chromatography coupled with OPA precolumn derivatization. Chagyo Kenkyu Hokoku, 77, [in Japanese]. Horie, H., & Kohata, K. (2000). Analysis of tea components by high-performance liquid chromatography and high-performance capillary electrophoresis. Journal of Chromatography A, 881, Juneja, L. R., Chu, D. C., Okubo, T., Nagato, Y., & Yokogoshi, H. (1999). L-theanine A unique amino acid of green tea and its relaxation effect in humans. Trends in Food Science & Technology, 10, Kolbezen, M. J., Eckert, J. W., & Bretschneider, B. F. (1962). The microdetermination of amines with 2,4-dinitrofluorobenzene. Analytical Chemistry, 34, Li, B. Q., & Hu, H. M. (1989). Rapid determination of theanine in tea. Journal of Instrumental Analysis, 8, [in Chinese]. Liang, Y. R., Lu, J. L., Zhang, L. Y., Wu, S., & Wu, Y. (2003). Estimation of black tea quality by analysis of chemical composition and colour difference of tea infusions. Food Chemistry, 80, Lockhart, I. M. (1956). Paper chromatographic identification of the 2:4-dinitrophenylderivatives of aliphatic amines. Nature, 177, Nakagawa, M., & Anan, T. (1979). A simple determination method of total amino acids in tea. Chagyo Kenkyu Hokoku, 50, [in Japanese]. Otsuki, K., Kawabata, M., Taguchi, K., & Kokura, H. (1979). High performance liquid chromatography of free amino acids in green tea extract Studies on prelabeling with dansyl chloride and the separation of the dansylamino acids.. Kyoto-furitsu Daigaku Gakujutsu Hokoku, Rigaku, Seikatsu Kagaku, 30, [in Japanese]. Sakato, Y. (1950). The chemical constituents of tea: III. A new amide, theanine. Nippon Nogei Kagaku Kaishi, 23, [in Japanese]. Sanger, F. (1945). The free amino groups of insulin. Biochemical Journal, 39, Sasaoka, K., Kito, M., & Inagaki, H. (1963). Biosynthesis of theanine in tea seedlings. Synthesis of theanine by homogenate of tea seedlings. Agricultural and Biological Chemistry, 27, Selvendran, R. R., & Selvendran, S. (1973). The distribution of some nitrogenous constituents in the tea plant. Journal of the Science of Food and Agriculture, 24, Sun, S. W., Lin, Y. C., Weng, Y. M., & Chen, M. J. (2006). Efficiency improvements on ninhydrin method for amino acid quantification. Journal of Food Composition and Analysis, 19, Takeo, T. (1980). Ammonium-type nitrogen assimilation in tea plants. Agricultural and Biological Chemistry, 44, Takeo, T. (1981). Nitrogen metabolism pertaining to biosynthesis of theanine in tea plants. Japan Agricultural Research Quarterly, 15, Thippeswamy, R., Gouda, K. G. M., Rao, D. H., Martin, A., & Gowda, L. R. (2006). Determination of theanine in commercial tea by liquid chromatography with fluorescence and diode array ultraviolet detection. Journal of Agricultural and Food Chemistry, 54, Tsushida, T., & Takeo, T. (1983). High-performance liquid chromatographic determination of theanine in tea by precolumn fluorescence derivatization with o-phthalaldehyde. Chagyo Gijutsu Kenkyu, 64, [in Japanese]. Tsushida, T., & Takeo, T. (1984). Occurrence of theanine in Camellia japonica and Camellia sasanqua seedlings. Agricultural and Biological Chemistry, 48, Wang, Z. N. (1984). Tea biochemistry. Protein and amino acids in tea plant: Varieties and distribution of amino acids in tea plant, Beijing: Chinese Agricultural Press. pp [in Chinese]. Ying, Y., Ho, J. W., Chen, Z. Y., & Wang, J. (2005). Analysis of theanine in tea leaves by HPLC with fluorescence detection. Journal of Liquid Chromatography & Related Technologies, 28, Zhu, Q., Shi, Z., Tong, J., & Ren, C. (2001). HPLC determination of free amino acid in instant green tea. Journal of Tea Science, 21, [in Chinese].