Food Chemistry () 66 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Analytical Methods Analysis of free ao acids in Chinese teas and flower of tea plant by high performance liquid chromatography combined with solid-phase extraction Lin Wang a, Renjie Xu a, Bing Hu a, Wei Li a, Yi Sun a, Youying Tu b, Xiaoxiong Zeng a, * a College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, China b Department of Tea Science, Zhejiang University, Hangzhou, Zhejiang, China article info abstract Article history: Received 8 December Received in revised form March Accepted May Keywords: Tea Free ao acids Derivatisation HPLC-DAD Solid-phase extraction An effective liquid chromatographic method that involved precolumn derivatisation with o-phthaladehyde combined with solid-phase extraction has been developed for the deteration of free ao acids in tea. Firstly, tea infusion was treated by C8 cartridge before derivatisation, resulting in great improvement of separation by Zorbax Eclipse XDB-C 8 column. Then, the contents of free ao acids in Chinese green, black, and Oolong teas and the flower of plant Camellia sinensis have been detered. The results showed that theanine was the most abundant ao acid in teas, and green tea contained much higher amounts of free ao acids than fermented ones. While the contents and composition of free ao acids in tea flower were quite different from those of teas. The tea flower contained much higher content of free ao acids. Furthermore, although theanine was the most abundant ao acid in tea flower as tea, histidine became the second one. Ó Elsevier Ltd. All rights reserved.. Introduction Tea is one of the most popular and widely consumed beverages in the world because of its refreshing taste, attractive aroma, and potential healthy benefits (Kuo et al., ). It is made from the leaves of the plant Camellia sinensis (L.). Generally, tea can be broadly classified according to the production method as unfermented tea (green tea), semi-fermented tea (Oolong tea), fully fermented tea (black tea) or post-fermented tea (pu-erh tea) (Zhao, Chen, & Huang, 6). Black tea is consumed worldwide, while green and Oolong teas are consumed mainly in Asia and North Africa. Nowadays, a lot of epidemiological and preclinical studies have demonstrated that drinking tea may reduce the risk of cancer and cardiovascular disease (Khan & Mukhtar, 7; Yang, Maliakal, & Meng, ). Moreover, other biological functions of tea have also been reported, such as anti-inflammation, anti-oxidation, anti-allergy, and anti-obesity (Fujimura, Tachibana, & Yamada, ; Khan & Mukhtar, 7). These beneficial effects have been attributed to the presence of tea compounds such as polyphenols, ao acids, vitas, carbohydrates, and purine alkaloids (Bolling & Chen, ). Theanine, c-glutamylethylamide or -N-ethyl glutae, is a non-protein ao acid that was first discovered in tea leaves (Sakato, ). It is the main free ao acid in teas, representing as much as % of the total ao acids in black tea and % of * Corresponding author. Fax: +86 867. E-mail address: zengxx@njau.edu.cn (X. Zeng). 8-86/$ - see front matter Ó Elsevier Ltd. All rights reserved. doi:.6/j.foodchem...6 the dry weight of green tea (Hara, Luo, Wikramasinghe, & Yamanishi, ). It not only plays an important role in the characteristic flavour and delicate taste of tea but also shows many biological effects. It involves in many biological activities such as promoting relaxation, inhibiting caffeine s negative effects, reducing blood pressure, and enhancing anti-tumor activity (Kimura, Ozeki, Juneja, & Ohira, 7; Sugiyama & Sadzuka, ; Yamada & Terashima, ). Moreover, it has been reported to have physiological activities including neuroprotection and anti-obesity (Cho et al., 8; Egashira et al., ; Zheng et al., ). Consequently, there is a demand for rapid and effective analytical methods for the analysis of free ao acids, which should be suitable across a wide range of research and practical applications. A number of methods have been developed to detere the presence of various ao acids in teas. The total contents of ao acids in teas can be detered by the ninhydrin or,-dinitrofluorobenzene colorimetric method (Chen, Chen, Zhang, & Wan, ). As for the analysis of free ao acid compositions in teas, various chromatographic methods including high performance liquid chromatography (HPLC), capillary electrophoretic and anion exchange chromatography have been reported (Alcazar et al., 7; Aucamp, Hara, & Apostolides, ; Ding, Yu, & Mou, ; Ohtsuki, Kawabata, Kokura, & Taguchi, 87; Pongsuwan et al., 8; Syu, Lin, Huang, & Lin, 8; Thippeswamy, Gouda, Rao, Martin, & Gowda, 6; Ying, Ho, Chen, & Wang, ). Due to the lack of a suitable chromophore, it is necessary to label the ao acids by using labeling reagent, such as -dimethylaoazobenzene- -sulfonyl chloride (dabsyl chloride), o-phthaladehyde 转载
6 L. Wang et al. / Food Chemistry () 66 (OPA), and phenylisothiocyanate. In such a case, it involves derivatisation with precolumn or postcolumn and detection by fluorescence or diode array detector (DAD). However, clear chromatograms are not obtained when the analysis of the composition of free ao acids in Chinese green teas was attempted, according to the reported methods by use of OPA as derivative reagent. In fact, tea infusion is usually prepared using distilled water. Accordingly, a number of other water-soluble extracts especially tea catechins are extracted out together with the free ao acids. They may affect the derivatisation or separation of the ao acids by HPLC. Therefore, it should be necessary to treat tea infusion before derivation by liquid liquid or solid-phase extraction (SPE). As we know, SPE is becog more used because it is rapid, economical, and sensitive. In addition, different cartridges with a great variety of sorbents can be used (del Alamo, Casado, Hernandez, & Jimenez, ; Perez-Magarino, Ortega-Heras, & Cano-Mozo, 8). However, there is little information about the application of SPE for the sample preparation in the analysis of free ao acids in tea. In this report, therefore, we introduced SPE combined with HPLC- DAD for the deteration of free ao acids including alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), serine (Ser), theanine (Thea), threonine (Thr), tryptohan (Trp), and tyrosine (Tyr) in Chinese teas. In addition, the contents of free ao acids in the flower of plant Camellia sinensis (L.) have also been analyzed by using the developed method.. Materials and methods.. Chemicals and regents Standards of ao acid, Ala, Arg, Asn, Asp, Gly, Ile, Leu, Lys, His, Met, Phe, Ser, Thr, Glu, Trp and Tyr, were purchased from Shanghai Kayon Biological Technology Co., Ltd. (Shanghai, China). A standard of Thea was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Standards of ( )-epicatechin (EC), ( )-epicatechin gallate (ECG), ( )-epigallocatechin (EGC), and ( )-epigallocatechin gallate (EGCG) were purchased from Funakoshi Co., Ltd. (Tokyo, Japan). OPA was obtained from Sigma (St. Louis, MO, USA). Hydrochloric acid, boric acid, formic acid, disodium phosphate and b-mercaptoethanol were obtained from Sinopharm Chemical Regent Co., Ltd (Shanghai, China). HPLC grade of acetonitrile and methanol were purchased from Hanbon Science and Technology (Jiangsu, China). The Sep-Pak-C8 cartridge was purchased from Waters Co., Ltd. (Milford, USA)... Tea samples and preparation of tea infusion In the present study, green teas were purchased from Hunan, Hubei, and Fujian in China, respectively. Black teas were obtained from Qimen Tea Factory (Anhui, China). Oolong teas and were purchased from Guangdong province in China, whereas Oolong tea was obtained from Taiwan, China. Firstly, all of these teas were ground into tea powders by a mill. Then, tea infusions were prepared as follows:. g of tea powder was extracted with ml of distilled water at C for. After the infusion was cooled to room temperature, it was made up to ml with distilled water and filtered through a. lm nylon filter membrane... Treatment of tea infusion by solid-phase extraction The SPE cartridges were conditioned by rinsing with ml of methanol and ml of distilled water. The tea infusion (. ml) was loaded onto the conditioned cartridge, and the retained compounds were eluted with. ml of % ethanol. The resulted elute was collected and evaporated to dryness in a rotary vacuum evaporator (Heidolph, Germany). Then, the residue was dissolved in. ml distilled water and filtered through a. lm nylon filter membrane before its precolumn derivatisation with OPA... Precolumn derivatisation with OPA The derivatisation with OPA was carried out according to the method reported with some modifications (Alcazar et al., 7; Thippeswamy et al., 6). Briefly, a 7 ll aliquot of tea infusion or standard ao acid solution was mixed with ll of OPA solution and incubated at ± C for exactly. Then, the reaction mixture was immediately used for HPLC analysis. The derivatisation solution was freshly prepared everyday as following: five milligrammes of OPA dissolved in. ml of methanol was added to. ml of. M boric acid/borate buffer (ph.), followed by addition of ll of b-mercaptoethanol... HPLC-DAD analysis The deteration of ao acids were performed by using an Agilent series HPLC system (Agilent Technologies, CA, USA), consisting of a model G7A degasser, a model GA quatpump, a model G6A column oven, and a model GB DAD. An Agilent ChemStation was used for instrument control and data acquisition. The separation was completed on a Zorbax Eclipse XDB-C 8 column (.6 mm, lm, Agilent). The temperature of column oven was set at C. The mobile phase consisted of methanol/acetonitrile/water (//, A) and phosphate buffer (ph 7., B). Elution was performed with a linear gradient as shown in Table. The flow rate was. ml/. The DAD was set at 8 nm to monitor the derivatised ao acids. The injection volume was ll..6. Validation of HPLC-DAD method HPLC-DAD validation tests were performed for accuracy, selectivity, linearity range, limit of detection (LOD), and limit of quantification (LOQ). The accuracy of the HPLC-DAD method was assessed by recovery experiments. Known amounts of standards of ao acids were added to tea infusion. The recovery was calculated by comparing the amount measured to that added. The selectivity criterion for an assay method is that the analyte peaks will have a chromatographic baseline with a suitable resolution from all of the other sample components. Calibration curves were constructed over five different concentrations. Each standard was analyzed in triplicate. LOD and LOQ were calculated as three and times, respectively, the ratio between the standard deviation of the y-intercept of the regression line and the average slope of the regression lines. Table Scheme of elution gradient for HPLC-DAD analysis. Time () Solvent A (%) Solvent B (%) 8 8 76.. 7.8. 7. 8 7 6
L. Wang et al. / Food Chemistry () 66 6.7. SPE method validation To demonstrate that SPE is an efficient way to deal with tea infusion, two special experiments were designed. Firstly, SPE accuracy experiment and recovery experiments were performed. A known quantity of standard ao acid solution (. ml) was loaded onto the conditioned cartridge, and the treatment of tea infusion was carried out by SPE. The obtained solution was concentrated, filtered, derivatised, and analyzed by HPLC-DAD. The recoveries were calculated by comparing the amount measured to that loaded. Secondly, SPE verify experiment was carried out. Tea infusion (. ml) was applied to conditioned SPE cartridge and the resulting elute was collected as fraction (F). Then the cartridge was washed by % ethanol (. ml), % ethanol (. ml), % ethanol (. ml), and 8% ethanol (. ml), respectively, and the corresponding elutes were collected as fraction (F), fraction (F), fraction (F), and fraction (F), respectively. All fractions were evaporated to dryness in a rotary vacuum evaporator, dissolved in a known volume (. ml) of distilled water, and filtered through a. lm nylon filter membrane, respectively. The contents of tea catechins in F F were analyzed by HPLC according to our previous reported method (Hu et al., ). Briefly, the fractions were separated by a TSKgel ODS-Z column (.6 mm, lm, Tosoh). The temperature of column oven was set at C, and the DAD was set at 8 nm. The mobile phase consisted of formic acid solution (ph., A) and methanol (B). Elution was performed with a linear gradient as follows:, A from 8% to %, B from 8% to 6%. System flow rate was. ml/. The contents of EC, EGC, ECG, and EGCG were calculated by the calibration curves as described in our previous report (Hu et al., ). In addition, F and F was mixed in equal volume to afford sample (S), and then in a similar manner, F, F, and F was added into S, respectively, affording sample (S), sample (S), and sample (S). And S S were derivatised with OPA and analyzed by HPLC-DAD as mentioned above, respectively.. Results and discussion.. Development and validation of HPLC-DAD analytical method HPLC separation of mixture of standard ao acids, Ala, Arg, Asn, Asp, Glu, Gly, Ile, Leu, Lys, His, Met, Phe, Ser, Thea, Thr, Trp, and Tyr, was achieved using a Zorbax Eclipse XDB-C 8 column in by a linear gradient elution of methanol/acetonitrile/water (//) and phosphate buffer (ph 7.) as shown in Table. The representative chromatogram of standard ao acids is shown in Fig.. Notably, the peaks showed resolutions higher than.8 for all of the detered compounds. For infusions of green, black, and Oolong teas treated by SPE, elution profiles of Ala, Arg, Asn, Asp, Glu, Gly, Ile, Leu, Lys, His, Met, Phe, Ser, Thea, Thr, Trp, and Tyr were similar to those observed for their corresponding standards as shown in Fig.. Therefore, peak identities in tea infusions were assigned based on comparison of peak retention times (t R ) and online DAD spectra of the authentic standards. The mean peak areas from triplicate HPLC-DAD analyses of a range of solution concentrations were used to detere calibration curves for each standard, which was defined as the leastsquares regression line relating absorbance peak area at 8 nm to sample concentration. We found that the coefficients of deteration (R ) were >. for calibration curves of all standards. The values of LOD, LOQ, and recovery rates were summarised as shown in Table. LODs were in the range of. 8. ng. Met and Ile had the highest LOD at 8. ng, following was Phe at. ng, while Ser had the lowest LOD at. ng. LOQ values were in the range of.8.7 ng, Met had the highest LOQ at.7 ng, and Ser had the lowest LOQ at.8 ng. For recovery rates, they ranged between 8.8% and 7.%... SPE treatment of tea infusion As shown in Fig., the mixture of 7 ao acid standards was well separated by the developed HPLC method. However, a clear chromatogram was not obtained for the direct derivatisation of tea infusion with OPA. The baseline was not steady and several peaks could not be separated effectively, and identified in the chromatogram of green tea infusion without SPE treatment as shown in Fig. a. Obviously, those peaks were not the ao acids that have been identified by the standards. In fact, similar phenomena have been found in previous studies (Alcazar et al. 7; Syu et al., 8). It might be caused by other compounds present in tea infusion. As we know, tea contains many natural polyphenols that more than 6 polyphenolic components are identified from green teas and fermented teas by Lin, Chen, and Harnly (8). Among them, gallic acid and tea catechins, mainly EC, ECG, EGC, and EGCG are the major functional components. Furthermore, water is often used to prepare tea infusion (Alcazar et al., 7; Pongsuwan et al., 8; Syu, Lin, Huang, & Lin, 8; Thippeswamy et al., 6). Accordingly, a lot of tea polyphenols are extracted out together with the free ao acids. We assumed that the phenolic compounds in tea infusion would be the main factor 6 8 7 6 7 Fig.. A typical HPLC-DAD chromatogram of 7 ao acids derivatised with OPA., Asp;, Glu;, Asn;, Ser;, His; 6, Arg; 7, Gly; 8, Thr;, Ala;, thea;, Tyr;, Met;, Trp;, Phe;, Ile; 6, Leu; and 7, Lys.
6 L. Wang et al. / Food Chemistry () 66 a 7 8 6 7 compounds in tea infusion would be the main factor affecting the analysis of free ao acids. In order to explain the role of SPE in the treatment, the SPE verification experiments were carried out. During the treatment of tea infusion by SPE, different fractions were collected as mentioned in experimental section, and the contents of major tea catechins and free ao acids were analyzed by HPLC-DAD respectively. We found that there were lower contents of tea catechins in F and F, while F and F contained large amounts of tea catechins as shown in Table and Fig.. The results demonstrated that the absorbed phenolic compounds in C8 car- b 6 8 6 7 c 6 8 6 7 Fig.. Representative elution profiles of tea infusions and flower of Camellia sinensis. (a) Green tea infusion with SPE treatment; (b) black tea infusion with SPE treatment; (c) Oolong tea infusion with SPE treatment; (d) the green tea infusion (without SPE treatment) and S S;(e) extract of tea flower (without SPE treatment); (f) extract of tea flower with SPE treatment. Refer to Fig. for peak identities. S S refer to the SPE verify experiment in experimental section. affecting the analysis of free ao acids. Therefore, we tried to use SPE to treat tea infusion before its derivatisation with OPA in the analysis of free ao acids. C8 cartridges have been the most widely used in phenolic compound separation, affording high concentration and purity of phenolic compounds (del Alamo et al., ; Perez-Magarino et al., 8). In the present study, we selected Oasis HLB cartridge for the remove of phenolic compounds present in tea infusions. As shown in Fig., the chromatograms were much better than those without SPE treatment. The results suggested that the phenolic
L. Wang et al. / Food Chemistry () 66 6 d 7 8 6 7 S S S S Original-green tea e 7 8 7 f 7 8 7 Fig. (continued) tridge was easily eluted out by % or % ethanol, while only a small part was washed out from the cartridge by using % ethanol solution. When the mixtures (S S) of different fractions were reacted with OPA and analyzed by HPLC-DAD, their chromatograms are shown in Fig. d. Comparing the contents of tea catechins in F F and the chromatograms of S S, it could be easily found that the higher concentration of tea catechins, the more unsteady chromatogram. Therefore, the phenolic compounds in tea infusion could affect the separation and deteration of free ao acids. Fortunately, the phenolic compounds in tea infusion could be effectively removed by the developed procedure by using C8 cartridge. In order to detere the contents of free ao acids in tea, we investigated whether the ao acids were absorbed or not by the C8 cartridge. As shown in Table, most ao acids were not retained in the cartridges, affording high recovery rates. However, several basic and polar ao acids, including Arg, Tyr, Trp, Phe, and Lys, were mostly adsorbed by the cartridge and could not be eluted out by water. In order to improve the recovery rates of those ao acids, we tried to use % ethanol solution instead of water to wash the cartridge. As a result, the recovery rates were improved (Table ), those of Arg, Tyr, Trp, Phe, and Lys increased from.%,.%,.%,.6%, and 8.% to 8.%,.%,.6%,.%, and 8.%, respectively. Since only a small part of the phenolic
6 L. Wang et al. / Food Chemistry () 66 Table Sensitivity and recovery for the deteration of ao acids and recovery for SPE treatment. Ao acid LOD (ng) LOQ (ng) Recovery (%) HPLC a SPE-W b SPE-W-E c Asp 7... 8.. Glu 8. 7.6..6 7. Asn 6..6...7 Ser..8. 6.. His 7... 8. 8. Arg 8. 7. 7.. 8. Gly 8.7 6. 7.8. Thr 6.8 6 8.8. 7.6 Ala 8. 6. 6. 7.. Thea. 7. 8. 7. Tyr. 8..6.. Met 8..7..7 8.8 Trp 8. 6. 6...6 Phe. 78. 6..6. Ile 8... 88.6. Leu.6. 6. 7. 8. Lys..8. 8. 8. a The recovery rates of the method HPLC-DAD, which was calculated by the results from the added standard ao acids. b The recovery rates of the standard ao acids which was eluted by water during the SPE procedure. c The recovery rates of the standard ao acids which was eluted by water and % ethanol during the SPE procedure. Table The contents of EC, EGC, ECG, and EGCG in F F. Components F F F F F EGC ND a.. ND ND EGCG ND.6.7. ND EC ND ND.7.6 ND ECG ND ND.6.6. Total amount ND..7.7. a ND: not detected. compounds was washed out from the cartridge by using % ethanol solution as mentioned above, % ethanol solution was used as the washing solution in the treatment of tea infusion by SPE... Deteration of free ao acids in Chinese teas Using this HPLC combined with SPE analytical method, the contents of free ao acids in green, black, and Oolong teas were detered. The results are shown in Fig. and Table. Notably, Thea was the most abundant ao acid and accounted for more than % of the total ao acids in teas. For green tea, Thea content varied from to lg/g. It was much higher than that of black tea or Oolong tea ( 6 lg/g). Furthermore, a clear corresponding relationship between the content of ao acid and the elaboration process of tea could be observed. Higher total amounts of free ao acids were present in green teas (6 68 lg/g). In addition, the levels of Asp, Glu, and Ser in green teas were much higher than those in Oolong and black teas. On the contrary, Ala was present only in fermented teas (..8 lg/g). The results are general in agree with those of previous reports (Alcazar et al., 7; Thippeswamy et al., 6). Green tea (non-fermented), Oolong tea (partially fermented), and black tea (fully fermented) are the three major commercial types of tea. Green tea is derived directly from inactivating by steag or microwave and drying the fresh tea leaves, thus, the chemical composition of green tea is very similar to that of fresh tea leaves. It contains appreciable amounts of free ao acids. For Oolong and black teas, most of tea catechins are enzymatic oxidised and polymerised during the fermentation process. Accompanying the process, the free ao acids in tea leaves may take great changes. Accordingly, Oolong and black teas contain less free ao acids compared with green tea. Although the ao acids contents of various teas were diverse even in the same kind, our results also demonstrated that Ala, Asp, Glu, Ser, and Thea were good discriating parameters. Consequently, the free ao acids can be considered as good chemical descriptors to differentiate green, black, and Oolong teas... Deteration of free ao acids in the flower of tea plant It has been reported that the flower of plant Camellia sinensis contains comparable tea catechins (Lin, Wu, & Lin, ; Yang, Jie, He, & Tu, 7). However, there is little information on free ao acids of tea flower. Therefore, the contents of free ao acids in tea flower were analyzed by the developed procedure. It can be seen from Fig. c and d that improvement in the HPLC chromatography was achieved through the treatment of tea flower extraction by SPE. It suggests that the phenolic compounds in tea flower may affect the analysis of free ao acids. Meanwhile, the contents and composition of free ao acids in tea flower were quite different from those of tea (Table ). Firstly, the tea flower contained much higher content (88 lg/g) of free ao acids. Secondly, although Thea was still the most abundant (76 lg/g) ao acid in tea flower as tea, His became the second one (8 lg/g), which was much higher than that of tea. In addition, Tyr, Phe, Ile, Leu and Lys were trace in tea flower. The results.8 Tea Catechins (mg).6.. F F F F F Fractions Fig.. The contents of tea catechins in the fractions of the SPE eluate. F F refer to the SPE verify experiment in experimental section.
L. Wang et al. / Food Chemistry () 66 6 Table The contents of free ao acids in Chinese teas a and flower of tea plant b. 6 7 8 Asp 77 ± 6.8 c 8 ± 8. 6 ±. 7. ±. 6. ±.7 ±. 6 ± 7.8 ±. 68 ±. Glu ±. 8 ± 7.8 6 ± 7. ±. ±.8 8 ± 6. 6 ± 8.6 ±.6 ±.8 Asn ± 6. 7 ± 86.7 6 ±.7 8 ±. ± 6.6 6.7 ±.. ±.8 ±. ±.7 Ser 8. ±. ± 6.7 6 ± 6. 8. ±. 67. ± 7.. ±.7.8 ±..7 ±.8 ±.8 His 7 ±. 8 ±. 6 ± 7.8 8 ± 7. ±.. ± 7.6. ±.7 8. ±. 8 ±.6 Arg ±. ±. ±. 66 ±. 7. ±. 66.6 ±.8. ±.87.8 ±.78 ND d Gly 6. ±. ND.7 ±. ND ND.78 ±. ND ND. ±.7 Thr 6.8 ±.7 7. ±. 6. ± 8.. ±..8 ±. 8.7 ± 6.7.7 ±..8 ±. 8. ±.8 Ala ND ND ND.8 ±.7. ± 7.8. ± 7. 8.7 ±.7. ±.8 8 ±.6 Thea ±.8 ±. ±. 6 ±. 88 ± 8. 87 ±. 87 ± 6.6 ±. 76 ± 8. Tyr.6 ± 8. 6. ±.6 6. ± 6.7 7.7 ±. 6. ±.. ±..7 ±.. ±.. ±. Met. ±. 7. ±.. ±.78 7. ±. 7.6 ±.87 7.6 ±.. ±.6.6 ±.. ±. Trp 6. ± 6.6 6. ± 8.8 6. ±. 7. ±.8. ±..8 ± 8.77.6 ±.. ±.8 8. ±. Phe 8. ± 8.. ±.8. ±. 6. ±.78 6. ±.. ±..8 ±.7.8 ±. Trace Ile 8.8 ±.. ± 6..8 ±..8 ± 7.7. ±.. ± 7. 8. ±.6 7.8 ±. Trace Leu. ±. 7.6 ±.8 7. ±.7 8. ±.7 7. ±..7 ±.67. ±.8 6.7 ±.6 Trace Lys. ±.. ± 6.8. ±. 6.6 ±.8 6. ±.7. ±.. ±.7 8.8 ±.8 Trace Total 6 6 68 87 7 8 88 a Chinese teas ( for green teas, and for black teas, and 6 8 for Oolong teas). b Flower of tea plant,. c Value (lg/g) = mean ± SD (n P ). d ND: not detectable. suggested that tea flower might be suitable for making alternative tea beverage.. Conclusion In summary, an effective method to detere the contents of free ao acids in tea and the flower of tea plant has been successfully developed by using HPLC-DAD combined with SPE. Firstly, tea infusion was treated by SPE cartridge before derivatisation. We found that the separation by Zorbax Eclipse XDB-C 8 column was improved greatly. Based on the results of the SPE verify experiment, we presumed that the phenolic compounds in tea infusion might affect the separation and deteration of free ao acids. In addition, the recovery rates of Arg, Tyr, Trp, Phe and Lys were got improved from.%,.%,.%,.6% and 8.% to 8.%,.%,.6%,.% and 8.%, respectively, by using % ethanol solution instead of water to wash the cartridge. Furthermore, the contents of free ao acids in green, black, and Oolong teas and tea flower have been detered. The results showed that Thea was the most abundant ao acids in teas, and green tea contained much higher amounts of free ao acids than fermented ones. The contents and composition of free ao acids in tea flower were quite different from those of teas. The tea flower contained much lower content of free ao acids. In addition, although Thea was still the most abundant ao acid in tea flower as tea, His became the second one. Acknowledgements This work was supported by a grant-in-aid from 86 Program, Ministry of Science and Technology of China under 7AAZ and 7AA. References Alcazar, A., Ballesteros, O., Jurado, J. M., Pablos, F., Martin, M. J., Vilches, J. L., et al. (7). Differentiation of green, white, black, oolong, and Pu-erh teas according to their free ao acids content. Journal of Agricultural and Food Chemistry,, 6 6. Aucamp, J. P., Hara, Y., & Apostolides, Z. (). Simultaneous analysis of tea catechins, caffeine, gallic acid, theanine and ascorbic acid by micellar electrokinetic capillary chromatography. Journal of Chromatography A, 876,. Bolling, B. W., & Chen, C. Y. (). Tea and health: Preventive and therapeutic usefulness in the elderly. Current Opinion in Clinical Nutrition and Metabolic Care,, 8. Chen, L., Chen, Q., Zhang, Z., & Wan, X. (). A novel colorimetric deteration of free ao acids content in tea infusions with,-dinitrofluorobenzene. Journal of Food Composition and Analysis,, 7. Cho, H. S., Kim, S., Lee, S. Y., Park, J. A., Kim, S. J., & Chun, H. S. (8). Protective effect of the green tea component, L-theanine on environmental toxins-induced neuronal cell death. Neuro Toxicology,, 66 66. del Alamo, M., Casado, L., Hernandez, V., & Jimenez, J. J. (). Deteration of free molecular phenolics and catechins in wine by solid phase extraction on polymeric cartridges and liquid chromatography with diode array detection. Journal of Chromatography A,, 7. Ding, Y. S., Yu, H., & Mou, S. F. (). Direct deteration of free ao acids and sugars in green tea by anion-exchange chromatography with integrated pulsed amperometric detection. Journal of Chromatography A, 8, 7. Egashira, N., Hayakawa, K., Mishima, K., Kimura, H., Iwasaki, K., & Fujiwara, M. (). Neuroprotective effect of c-glutamylethylamide (theanine) on cerebral infarction in mice. Neuroscience Letters, 6, 8 6. Fujimura, Y., Tachibana, H., & Yamada, K. (). Lipid raft-associated catechin suppresses the FceRI expression by inhibiting phosphorylation of the extracellular signal-regulated kinase/. FEBS Letters, 6,. Hara, Y., Luo, S. J., Wikramasinghe, R. L., & Yamanishi, T. (). Special issue on tea. Food Reviews International,, 7. Hu, B., Wang, L., Zhou, B., Zhang, X., Sun, Y., Hong, Y., et al. (). Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins, three purine alkaloids, and gallic acid in tea by high-performance liquid chromatography with diode array detection. Journal of Chromatography A, 6,. Khan, N., & Mukhtar, H. (7). Tea polyphenols for health promotion. Life Science, 8,. Kimura, K., Ozeki, M., Juneja, L. R., & Ohira, H. (7). L-theanine reduces psychological and physiological stress responses. Biological Psychology, 7,. Kuo, K. L., Weng, M. S., Chiang, C. T., Tsai, Y. J., Lin-Shiau, S. Y., & Lin, J. K. (). Comparative studies on the hypolipidemic and growth suppressive effects of Oolong, black, pu-erh, and green tea leaves in rats. Journal of Agricultural and Food Chemistry,, 8 8. Lin, L. Z., Chen, P., & Harnly, J. M. (8). New phenolic components and chromatographic profiles of green and fermented teas. Journal of Agricultural and Food Chemistry, 6, 8 8. Lin, Y. S., Wu, S. S., & Lin, J. K. (). Deteration of tea polyphenols and caffeine in tea flowers (Camellia sinensis) and their hydroxyl radical scavenging and nitric oxide suppressing effects. Journal of Agricultural and Food Chemistry,, 7 8. Ohtsuki, K., Kawabata, M., Kokura, H., & Taguchi, K. (87). Simultaneous deteration of S-methylmethionine, vita U and free ao acids in extracts of green tea with HPLC-ao acid analyzer. Agricultural and Biological Chemistry,, 7 8. Perez-Magarino, S., Ortega-Heras, M., & Cano-Mozo, E. (8). Optimization of a solid-phase extraction method using copolymer sorbents for isolation of
66 L. Wang et al. / Food Chemistry () 66 phenolic compounds in red wines and quantification by HPLC. Journal of Agricultural and Food Chemistry, 6, 6 7. Pongsuwan, W., Bamba, T., Harada, K., Yonetani, T., Kobayashi, A., & Fukusaki, E. (8). High-throughput technique for comprehensive analysis of Japanese green tea quality assessment using ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS). Journal of Agricultural and Food Chemistry, 6, 7 78. Sakato, Y. (). The chemical constituents of tea III. A new amide theanine. Journal of Agricultural and Food Chemistry,, 6 67. Sugiyama, T., & Sadzuka, Y. (). Theanine and glutamate transporter inhibitors enhance the antitumor efficacy of chemotherapeutic agents. Biochimica et Biophysica Acta, 6, 7. Syu, K. Y., Lin, C. L., Huang, H. C., & Lin, J. K. (8). Deteration of theanine, GABA, and other ao acids in green, oolong, black, and Pu-erh teas with dabsylation and high-performance liquid chromatography. Journal of Agricultural and Food Chemistry, 6, 767 76. Thippeswamy, R., Gouda, M. K. G., Rao, D. H., Martin, A., & Gowda, L. R. J. (6). Deteration of theanine in commercial tea by liquid chromatography with fluorescence and diode array ultraviolet detection. Journal of Agricultural and Food Chemistry,, 7 7. Yamada, T., & Terashima, T. (). Theanine, c-glutamylethylamide, a unique ao acid in tea leaves, modulates neurotransmitter concentrations in the brain striatum interstitium in conscious rats. Ao Acids, 6, 7. Yang, C. S., Maliakal, P., & Meng, X. (). Inhibition of carcinogenesis by tea. Annual Review of Pharmacology and Toxicology,,. Yang, Z. Y., Jie, G., He, P. M., & Tu, Y. (7). Study on the antioxidant activity of tea flowers (Camellia sinensis). Asia Pacific Journal of Clinical Nutrition, 6, 8. Ying, Y., Ho, J. W., Chen, Z. Y., & Wang, J. (). Analysis of theanine in tea leaves by HPLC with fluorescence detection. Journal of Liquid Chromatography and Related Technologies, 8, 77 77. Zhao, J. W., Chen, Q. S., & Huang, X. Y. (6). Qualitative identification of tea categories by near infrared spectroscopy and support vector machine. Journal of Pharmaceutical and Biomedical Analysis,, 8. Zheng, G., Bamba, K., Okubo, T., Juneja, L. R., Oguni, I., & Sayama, I. K. (). Effect of theanine, c-glutamylethylamide, on bodyweight and fat accumulation in mice. Animal Science Journal, 76, 7.