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1 Hong Kong Baptist University HKBU Institutional Repository HKBU Staff Publication 0 Comparison of ten major constituents in seven types of processed tea using HPLC-DAD-MS followed by principal component and hierarchical cluster analysis Tao Yi Hong Kong Baptist University, yitao@hkbu.edu.hk Lin Zhu Hong Kong Baptist University Wan-Ling Peng Hong Kong Baptist University Xi-Cheng He Hong Kong Baptist University Hong-Li Chen Lanzhou University See next page for additional authors This document is the authors' final version of the published article. Link to published article: Recommended Citation Yi, Tao, Lin Zhu, Wan-Ling Peng, Xi-Cheng He, Hong-Li Chen, Jie Li, Tao Yu, Zhi-Tao Liang, Zhong-Zhen Zhao, and Hu-Biao Chen. "Comparison of ten major constituents in seven types of processed tea using HPLC-DAD-MS followed by principal component and hierarchical cluster analysis." Food Science and Technology., Part (0): -0. This Journal Article is brought to you for free and open access by HKBU Institutional Repository. It has been accepted for inclusion in HKBU Staff Publication by an authorized administrator of HKBU Institutional Repository. For more information, please contact repository@hkbu.edu.hk.

2 Authors Tao Yi, Lin Zhu, Wan-Ling Peng, Xi-Cheng He, Hong-Li Chen, Jie Li, Tao Yu, Zhi-Tao Liang, Zhong-Zhen Zhao, and Hu-Biao Chen This journal article is available at HKBU Institutional Repository:

3 *Manuscript Click here to view linked References Comparison of ten major constituents in seven types of processed tea using HPLC-DAD-MS followed by principal component and hierarchical cluster analysis Tao Yi a,*,, Lin Zhu a,, Wan-Ling Peng a, Xi-Cheng He a, Hong-Li Chen b, Jie Li c, Tao Yu a, Zhi-Tao Liang a, Zhong-Zhen Zhao a, Hu-Biao Chen a,* Affiliation a School of Chinese Medicine, Hong Kong Baptist University, Hong Kong Special Administrative Region, China. b Department of Chemistry, Lanzhou University, Lanzhou 0000, China. c Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 00 West th Avenue, Columbus,, USA. * Corresponding Author School of Chinese Medicine, Hong Kong Baptist University, Hong Kong Special Administrative Region, P. R. China. Tel.: + 0, + 00; fax: +, +. address: yitao@hkbu.edu.hk (T. Yi), hbchen@hkbu.edu.hk (H. B. Chen). 0 These authors contributed equally to this work. Abbreviations: GT, green tea; YT, yellow tea; WT, white tea; OT, oolong tea; BT, black tea; APT, aged pu-erh tea; RPT, ripened pu-erh tea; PCA, principal component analysis; HCA, hierarchical cluster analysis.

4 ABSTRACT A new HPLC-DAD-MS method was developed to compare the major constituents in types of processed tea, namely green tea, yellow tea, white tea, oolong tea, black tea, aged pu-erh tea and ripened pu-erh tea. MS was used for identification in positive ion mode, and DAD was used for quantification at wavelength of nm. Ten components were simultaneously determined in tea samples representing processing types, and then principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to distinguish and classify between the samples. The results demonstrate that the contents of the major constituents significantly varied among the types of tea. Unique aspects of each type of processing were correlated with unique aspects of the chemistry of the tea. The types of processed tea were successfully divided into four categories based on our determination and chemometrics analysis. Our present method was adaptable for the comparative study of processed tea, which significantly contributes to discrimination and quality evaluation of teas. KEYWORDS: Processed tea, Major constituents, HPLC-DAD-MS, Principal component analysis, Hierarchical cluster analysis 0 Chemical compounds studied in this article Gallic acid (PubChem CID: 0); Theobromine (PubChem CID: ); (-)-Gallocatechin (PubChem CID: ); (-)-Epigallocatechin (PubChem CID: ); (+)-Catechin (PubChem CID: 0); Caffeine (PubChem CID: ); (-)-Epicatechin (PubChem CID: ); (-)-Epigallocatechin gallate (PubChem CID: 0); (-)-Gallocatechin gallate (PubChem CID: ); (-)-Epicatechin gallate (PubChem CID: 0)

5 0. Introduction Tea, next to water, is the second most popular beverage in the world (Shahidi, ). Tea contains multiple constituents, and catechins as well as alkaloids are considered to be the major bioactive components among these chemicals (Da Silva Pinto, 0; Wang et al., 0). It is well known that tea has extensive health benefits for humans, including anti-oxidation (He & Shahidi, ), anti-obesity (Sergent, Vanderstraeten, Winand, Beguin, & Schneider, 0), anti-viral (Zhong, Ma, & Shahidi, 0), cholesterol-lowering effect (Chan et al., ), and reducing the risks of cancer (Wu et al., 00; Zhong, Chiou, Pan, Ho, & Shahidi 0). Due to its powerful anti-oxidant and anti-microbial properties, tea extract has also been used as a natural preservative to increase shelf life of foods (Dong, Zhu, Li, & Li, 0; Oh, Jo, Cho, Kim, & Han, 0). In general, teas are processed before use. There are basically processing methods in China, producing the main varieties of tea to meet the needs of different consumers (Lu & Shen, 0; Zhou, Duan, Wu, & Si, 0). They are green tea (GT), yellow tea (YT), white tea (WT), oolong tea (OT), black tea (BT), aged pu-erh tea (APT) and ripened pu-erh tea (RPT). Their processing protocols are illustrated in Diagram. However, the processed teas are always confused, because few chemical characteristics of these teas have been described systematically. Insert Diagram here On the other hand, consumers have the opportunity of exposure to various teas, and they are eager to know their difference and how to distinguish between them. For tea producers and tea regulatory agencies, they also want to establish a specific quality standard based on

6 0 the characteristics of individual tea for quality assurance and quality control (QA & QC). Therefore, a comparative study of the major chemical constituents in various processed teas is urgently needed now. Recently, chemical analysis of tea has been carried out using TLC (Cimpoiu, Hosu, Seserman, Sandru, & Miclaus, 0), HPLC (Song, Li, Guan, Wang, & Bi, 0; Rahim, Nofrizal, & Saad, 0; Wang, Helliwell, & You, 000; El-Shahawi, Hamza, Bahaffi, Al-Sibaai, & Abduljabbar, 0) and UPLC-MS (Fraser et al., 0; Pongsuwan et al., 00). However, the existing studies mainly focus on the determination of a single or a few types of tea based on a few makers. Up to date, compositional data generated using the same extraction and the same validated methodology are still scarce, and comparative analysis of a comprehensive range of teas simultaneously by a single method has not been reported (Stodt & Engelhardt, 0). Inspired by the above-mentioned problem, in the present work, we aimed: (i) to develop a new HPLC-DAD-MS method for simultaneous determination of major components ( catechins, alkaloids and gallic acid) in tea samples produced by processing methods; (ii) to classify the tea samples by principal component analysis (HCA) and hierarchical cluster analysis (HCA) and, finally, (iii) to correlate the chemical composition of different teas with their processing methods. By obtaining a clearer view on chemical composition of types of processed tea, this study has a considerable significance for consumer, producer and quality control authorities concerned to teas.. Materials and methods

7 0.. Materials and reagents Seventy-four tea samples, namely: green tea (GT) samples, yellow tea (YT) samples, white tea (WT) samples, oolong tea (OT) samples, black tea (BT) samples, aged pu-erh tea (APT) samples and ripened pu-erh tea (RPT) samples were collected from China. Detailed information of these teas is listed in the Table S of the Supporting data. Gallic acid (GA), caffeine (CAF), theobromine (TBM), (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-gallocatechin gallate (GCG), (-)-epigallocatechin gallate (EGCG), and (-)-epicatechin gallate (ECG) standards were purchased from Phytomarker Ltd. (Tianjin, China). The purity of these chemical standards was no less than %. The chemical structures of standards are shown in Figure. Formic acid of analytical grade was purchased from Merck (Darmstadt, Germany). Acetonitrile of HPLC grade was purchased from Lab-scan (Bangkok, Thailand). Water was purified using a Milli-Q water system (Millipore; Bedford, MA, USA)... HPLC-DAD-MS analysis Insert Figure here An Agilent 0 high-performance liquid chromatography (HPLC) system with diode array detector (DAD), was used for quantitative analysis. Detection wavelength was set at nm. An Alltima C column ( μm,. 0 mm) was used for chromatographic separation at 0 o C. The mobile phase consisted of 0.% formic acid in water (A) and 0.% formic acid in acetonitrile (B) using a gradient program of % (B) in 0- min, and -0% in -0 min. The solvent flow rate was ml/min. For mass spectrometric determination, the HPLC-DAD system was hyphenated to an Agilent 0 quadrupole time-of-flight mass

8 spectrometry (MS) system by an electrospray ionization (ESI) interface (Santa Clara, CA, USA). The effluent from DAD was drained to the MS system with a split ratio of :. The conditions of MS analysis in positive ion mode were as follows: scan range, 0-00 m/z; drying gas (nitrogen), flow rate, L/min; gas temperature, 00 o C; capillary voltage, kv; fragmentor voltage, 0 V; nebulizer pressure, kpa. All operations and data analysis were controlled by Agilent MassHunter Workstation software version B Preparation of standard and sample solutions For the calibration, EGC, CAF, EGCG, GCG and ECG working solutions of -0 mg/l, C, EC and GC solutions of -0 mg/l, GA solutions of 0.- mg/l and TBM of 0.- mg/l were prepared in 0% methanol and stored at o C before use. Each tea sample was milled (ca. 0. mm); of the milled tea, 0. g was accurately weighted and transferred into a 0 ml conical centrifuge tube. The tea sample was extracted with 0 ml of water at the temperature of o C for min with intermittent shaking. The operations were repeated two times. Total extracts were combined in a 0-mL volumetric flask, which was filled up to the calibration mark with water. The extracts were then filtered through a 0. μm Millipore filter. An aliquot of μl solution was injected for HPLC-DAD-MS analysis. 0.. Assay validation and sample determination MS was used for identification in positive ion mode, and DAD was used for quantification at wavelength of nm. Linearity for standards was determined with seven data points over the concentration range of the working solutions. Repeatability was evaluated by six

9 injections of the sample solution (GT) within one day. Reproducibility was evaluated in intra- and inter-day assays of the tea sample GT. The stability test was performed by analyzing the sample solution (GT) over period of h. The relative standard deviation (RSD) was taken as the measures of precision, repeatability and stability. To determine the recoveries, sample GT spiked with standards at low, middle and high concentration levels in three replicates were extracted and analyzed. Recovery was calculated by dividing the amount of analyte found in the spiked sample by the sum of the amount originally found in the sample plus the amount spiked. All tea samples were analyzed using the described method... Data analysis The mean value and standard deviation (SD) of analytes was calculated from the experimental data. The significance (P < 0.0) between two sets of data was determined by unpaired t-test using the software package Prism version.0 (GraphPad Software, Inc., La Jolla, CA, USA). To classify and discriminate between the tea samples, principal component analysis (PCA) and hierarchical cluster analysis (HCA) was performed with SPSS for Windows version 0.0 (SPSS, Chicago, IL, USA). 0. Results and discussion.. Optimization of the sample extraction Brewing tea with hot water for a short while is the most popular way of tea drinking. Thus, extraction of tea with hot water was chosen in this study. Infusion period was chosen from to 0 min, and the results showed that the maximum release of analytes reached at min of

10 infusion, followed by a constant decrease due to the thermal instability of catechins. Extraction times was further optimized, the results showed that that exhaustive extraction could be achieved when 0. g tea sample powder was extracted with 0 ml water brewing for min with intermittent shaking, three times (Figure S). 0.. Optimization of the analysis conditions By comparing the HPLC chromatograms of tea extracts acquired at different wavelengths within the range 0 00 nm, and the corresponding UV absorption maxima for each standard compound, it was found that nm was more sensitive with lower interference. Therefore, nm was chosen as the determination wavelength. Different ratios of acetonitrile and water were further tried, until satisfactory resolutions for the analytes within 0 min were obtained. Compared to the existing reports (Stodt et al., 0), the present gradient elution condition presented the shortest analysis time for separation of ten analytes in tea by using HPLC. The typical chromatograms of seven teas at nm are shown in Figure A. Insert Figure here In order to further obtain a comprehensive view on chemical constituents in seven types of tea, the mass spectrometric conditions were optimized in both positive and negative ion modes. Results revealed that the positive ion mode was more sensitive. The typical total ion chromatograms (TICs) of seven types of tea are shown in Figure B. The results show that unambiguous identification of analytes under the optimized conditions was achieved (Figure S).

11 0.. Validation of the analysis method The validity of the method was well assessed, and the results are summarized in Table (details are listed in Table S to Table S of the Supporting data). For all analytes, a good linearity with R > 0. was achieved. Based on visual evaluation with a signal-to-noise ratio of about : and :, the LOD and LOQ of the analytes were found to be less than. and less than. ng, which were lower than the existing reports (Rahim et al., 0). The RSD values of reproducibility were reported within the range of 0. and.% for intra-day assays and 0. to.% for inter-day assays. The average recovery of the ten analytes ranged from.% to.%. The results of stability test suggested that the tea extract sample was stable in the experiment (RSD <.%). Based on these results we concluded that the overall analytical procedure is sensitive, precise and accurate, which is considered suitable for determination of tea samples. Insert Table here.. Comparison of the components in types of processed tea The tea samples, representing processing methods, were analyzed using the present method, and the results are summarized in Table. To clarify the differences between the various teas, we describe the characteristics of these processing methods, and then correlate the key process with the chemical composition of each type of tea. Insert Table here Green Tea (GT). The first step of processing GT is called green-killing ( Shaqing in Chinese), which is also the key process (Diagram ). Green-killing refers to quick application

12 0 of heat, either with steam, or by parching in hot pans, to halt oxidation and fix most of the chemical constituents of the tea leaves (Lu et al., 0; Zhou et al., 0). As shown in Table, it was found that EGCG (.0 mg/g) and CAF (. mg/g) were the two most abundant of the ten components in GT, and in fact GT possessed the most abundant EGCG and CAF of all the other types of tea. This finding may correlate with the short processing period of GT, and the degradation of catechins in tea was inhibited. Yellow Tea (YT). Yellowing process is unique to YT. Tea leaves after green-killing are allowed to be lightly heated (c.a. o C) in a closed humid container for appropriate time, which causes a distinctive yellowish-green hue of the tea leaves (Zhou et al., 0). And then, the other steps are as same as those of GT. As shown in Table, there was no significance in the contents of ten analytes between YT and GT (p > 0.0); the content of total catechins was. mg/g, which was comparable to the content of total catechins of. mg/g in GT. It was revealed that the similarity of chemical composition between GT and YT may be related to their similar processing methods (Diagram ). White Tea (WT). WT is prepared from the leaves of albino tea tree, and the leaves harvested are white. They are allowed to wilt for a brief time, and then baked dry. During the wilting process, the tea leaves are shielded from sunlight to keep them white and prevent the formation of pigments. As shown in Table, the contents of four components in WT, namely GA, GC, EGC and EC, were comparable to those in GT (p > 0.0); while the contents of other six components were significantly lower than those in GT (TBM, **, p < 0.0; C, CAF, EGCG, GCG and ECG, ***, p < 0.00). Overall, the total content of the ten components in WT was still lower than that in GT.

13 0 Oolong Tea (OT). OT is a type of a partially oxidized tea, produced by a unique process called blue-making ( Zuoqing in Chinese, Diagram ). Blue-making includes repeatedly tossing the leaves in baskets and stacking them indoors. The degree of oxidation of OT can be adjusted by increasing or reducing the cycles of blue-making (Lu et al., 0; Zhou et al., 0). Compared to the other teas (Table ), OT has two special characteristics in chemical composition. Firstly, OT possesses the highest content of EGC (. mg/g) and lowest content of CAF (. mg/g) of all the teas. Secondly, the ratio of total catechins/ total alkaloids in OT is the highest (up to.). These results indicate that blue-making can reduce levels of alkaloids and improve the content of EGC. Black Tea (BT). BT is a completely oxidized tea. The withered tea leaves undergo bruising through crushing or cutting to disrupt leaf cell structures, fully releasing the leaf juices and enzymes that activate complete oxidation (Diagram ). As shown in Table, the contents of all seven catechins (GC, EGC, C, EC, EGCG, GCG and ECG) in BT is significantly decreased compared to those in GT ( ***, p < 0.00). Although the content of CAF also decreased to. mg/g ( **, p < 0.0), CAF is still the most abundant component in BT. Interestingly, the content of GA in BT (. mg/g) is significantly increased compared to that in GT (.0 mg/g; ***, p < 0.00). The increased GA may be generated from the galloyl moiety of EGCG, GCG and ECG during the bruising stage of the processing. Aged Pu-erh Tea (APT). APT derives from GT that has undergone a natural aging process during storage at room temperature in normal humidity for a period of years (Zhou et al., 0). Compared to the GT, APT has a longer storage time before consumption. As shown in Table, storage did not affect the contents of alkaloids, TBM (. mg/g) and CAF (.

14 0 mg/g) in APT were almost equivalent to those in GT (. mg/g for TBM and. mg/g for CAF), respectively. Compared to the catechins in GT, three catechins in APT were decreased, namely EGC (. mg/g; *, p < 0.0), EGCG (. mg/g; ***, p < 0.00) and GCG (. mg/g; ***, p < 0.00), while three catechins were increased, namely C (. mg/g; **, p < 0.0), EC (. mg/g; ***, p < 0.00) and ECG (0.0 mg/g; ***, p < 0.00), and one catechin was unchanged, namely GC (. mg/g, p > 0.0). As with BT, the content of GA (. mg/g) in APT was significantly increased compared to that in GT (.0 mg/g; ***, p < 0.00). It is noteworthy that, the content (0.0 mg/g) of ECG in APT was the highest among all the types of teas; thus ECG is a unique component in the chemical composition of APT. Ripened Pu-erh Tea (RPT). RPT has undergone an accelerated oxidation process known as wet piling ( Wodui in Chinese, Diagram ). Freshly picked tea leaves are spread on trays, allowed to wilt, and then, sprayed with water. The trays are stacked, and stored in a controlled environment of kept at 0-0 o C by adjusting the humidity. Under these conditions, the speed and degree of oxidation is higher than APT. As shown in Table, we found that all catechins in RPT were significantly reduced ( ***, p < 0.00) compared to those in GT, and the contents of alkaloids in RPT was not affected by the process of wet piling (p > 0.0). The ratio of total catechins/ total alkaloids in RPT dropped to 0., which was comparable to BT (0.; p > 0.0). This finding indicates that RPT is the most oxidized of all the seven types of tea. In terms of other parameters, the contents as well as the ratio of the ten components in RPT were similar to those in BT... Classification of the types of processed tea Principal component analysis (PCA) and hierarchical cluster analysis (HCA) are two main

15 0 approaches in chemometrics, and they are widely used for the classification study in the field of food research (Yu, 00). PCA is a statistical data reduction method. It transforms the original set of variables to a new set of uncorrelated variables called principal components (PCs). By plotting the PCA scores, it is possible to visually assess similarities between samples and determine whether samples can be grouped. In our study, the initial eigenvalues were generated by inputting the contents of ten determined components in the tea samples to SPSS software. The cumulative percent variance (CPV) of three principal variables was found to be. % of the total variance, which meets the general requirements of CPV > 0%~% for PCA analysis (Liu, 00). Thus, the resulting data was plotted to produce a three-dimensional (D) graphic of PCA scores shown in Figure. Insert Figure here Hierarchical cluster analysis (HCA) involves a measurement of the similarity between objects to be clustered, and samples with the maximum similarities were clustered preferentially (Yi et al., 0). In this study, the ten determined components of the tea samples was inputted into SPSS as variables, between group average linkage method was applied to sort tea samples into groups, and rescaled distance was selected as measurement to obtain a HCA dendrogram shown in Figure. Insert Figure here As shown in the PCA graphic and HCA dendrogram, the tea samples representing the types of tea processing methods were clearly clustered in four main groups. From the results, it was shown that BT and RPT, the two completed oxidized teas, were clustered into group I. This finding revealed that both bruising for BT and wet piling for RPT during

16 processing leads to the completed oxidization, which contribute to the global similarity in the chemical composition of BT and RPT. The second group (group II) was constituted by the two unoxidized teas, YT and GT. The contents of the major constituents in the two types of teas are similar due to their similar processing methods. The third group (group III) is composed of OT and WT, the two partially oxidized teas. The key process, wilting in shield for WT and blue-making for OT, makes tea leaves slightly oxidized. Moreover, all of the OT samples and half of WT samples (WT, WT, WT and WT) originated in Fujian province of China. These reasons perhaps explain why OT and WT were clustered into a group in the PCA and HCA graphics. The fourth group (group IV) includes only APT. In the PCA and HCA graphics, all the APT samples were clustered away from other types of tea, visually representing the change in the chemical composition after processing (i.e., half of catechins reduced, the other half increased, and alkaloids unchanged). This change decided the chemical composition of APT was distinctive from other teas. Overall, the types of processed tea were successfully divided into four categories based on our determination and chemometrics analysis. 0. Conclusion In conclusion, a highly precise and accurate HPLC-DAD-MS method was developed to determine the major components of tea samples, which represented types of tea processing methods in China. Our present method is adaptable for the quality evaluation of tea for tea producers and regulatory authorities. By comparing the contents of the major components and correlation analysis, the unique aspects of each type of tea processing were

17 described systematically and correlated with the characteristics in the chemical composition. The types of tea processing methods were clearly clustered in four main groups. This study not only provides scientific information for consumers to distinguish different teas, but also advanced our knowledge about the effect of processing on the composition of tea.

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22 Table Legends for Tables Table Linearity, sensitivity, precision, accuracy and stability of the method. Table The contents of the ten components in seven types of tea.

23 Table Linearity, sensitivity, precision, accuracy and stability of the method. Repeata Reproducibility Linearity Recovery (mean, %, n =) Stability Peak RT Compo LOD LOQ bility (RSD, %, n =) (RSD, No. (min) nents Range (ng) (ng) (RSD, Equation R Intra- Inter- Low Middle High Average %, n =) (mg /L) %, n =) day day.0 GA 0.- y =. x TBM 0.- y =. x GC -0 y =.0 x EGC -0 y =. x C -0 y =. x CAF -0 y =. x EC -0 y =. x EGCG -0 y =.0 x GCG -0 y =. x ECG -0 y =. x

24 Table The contents of the ten components in seven types of tea. Component Contents a) (mg/g) GT ( b) ) YT () WT () OT () BT () RPT () APT () GA.0 ± 0.. ±.. ±.0 0. ± 0. *** c). ±. ***.0 ±.. ±. *** TBM. ± ± ± 0. ** 0. ± 0. ***.0 ± 0.. ± 0.. ± 0. GC.0 ±.. ±.. ±..0 ±. 0. ± 0.0 *** 0.0 ± 0. ***. ±. EGC. ±.0 0. ±.. ±.. ±. *** 0. ± 0. *** 0. ± 0. ***. ±. * C. ±.. ±..0 ± 0.0 ***.0 ± 0. *** 0. ± 0. *** 0. ± 0. ***. ±. ** CAF. ±.. ±.. ±. ***. ±. ***. ±. **. ±.. ±. EC. ±.. ±.. ±..00 ±. 0. ± 0. ***. ± 0. ***. ±. *** EGCG.0 ±.. ±.. ±. ***. ±. ***. ±.0 ***. ±. ***. ±.0 *** GCG. ±.. ±.0. ±. ***.0 ± 0. *** 0. ± 0. *** 0. ± 0. ***. ±. *** ECG. ±.. ±.0. ±.0 ***.0 ±. ***. ±. ***. ±. *** 0.0 ±. *** Total alkaloids (TA) Total catechins (TC) Ratio of TC/ TA a) The value is mean ± S.D. of samples from the same type of tea. b) The number of samples for each type of tea. c) *, p < 0.0; **, p < 0.0 and ***, p < 0.00 with respect to GT group.

25 Figure Legends for Figures Diagram. Illustration of the preparation of the seven types of Chinese tea ( the key process). Fig.. Chemical structures of the ten main components in tea. Fig.. Typical (A) UV chromatograms at nm and (B) TIC mass spectra in positive ion mode of seven processed Chinese teas and reference standards. (, GA;, TBM;, GC;, EGC;, C;, CAF;, EC;, EGCG;, GCG;, ECG; STs, reference standards). Fig.. D graphic of PCA scores by the ten quantified components in tea samples ( GT samples, YT samples, WT samples, OT samples, BT samples, APT samples and RPT samples). Fig.. HCA dendrogram of tea samples. ( GT samples, YT samples, WT samples, OT samples, BT samples, APT samples and RPT samples).

26 Natural aging Wet piling Aged pu-erh tea Ripened pu-erh tea Steaming Parching Rolling Forming Drying Green tea Sweltering Rolling Drying Yellow tea Wilting in shade Baking Light rolling Drying White tea Fresh tea leaves Sunned wilting Partial oxidation Tossing in basket Indoor stacking Blue-making Parching Baking Rolling Ball rolling Drying Firing Oolong tea Sunned wilting Light crushing Full oxidation CTC with full oxidation Rolling Drying Black tea WILTING BRUISING PRE-FERMN FIXATION (Green-killing) YELLOWING SHAPING DRYING POST-FERMN (Aging) PRODUCTS Diagram. Illustration of the preparation of the seven types of Chinese tea ( the key process).

27 O HO O N N O N N HO HN N N N O O Gallic acid (GA) Theobromine (TBM) Caffeine (CAF) HO O HO O HO O O O O O O O HO (-)-Epigallocatechin gallate (EGCG) HO (-)-Gallocatechin gallate (GCG) HO (-)-Epicatechin gallate (ECG) HO O HO O HO O HO O (-)-Gallocatechin (-)-Epigallocatechin (+)-Catechin (-)-Epicatechin (GC) (EGC) (C) (EC) Fig.. Chemical structures of the ten main components in tea.

28 x 0 GT A GT B x 0 x 0 x 0 x 0 YT WT OT Response Units vs. Acquisition Time (min) APT YT WT OT APT x 0 RPT RPT x 0 x 0 BT STs Response Units vs. Acquisition Time (min) BT STs Fig.. Typical (A) UV chromatograms at nm and (B) TIC mass spectra in positive ion mode of seven processed Chinese teas and reference standards. (, GA;, TBM;, GC;, EGC;, C;, CAF;, EC;, EGCG;, GCG;, ECG; STs, reference standards).

29 Group I Group II Group III Group IV Fig.. D graphic of PCA scores by the ten quantified components in tea samples ( GT samples, YT samples, WT samples, OT samples, BT samples, APT samples and RPT samples).

30 0 0 BT BT BT BT BT RPT RPT RPT RPT RPT RPT RPT RPT BT RPT BT RPT BT RPT YT YT GT YT GT GT GT GT GT GT GT GT GT GT GT GT GT GT YT YT GT GT YT OT OT OT OT OT OT OT OT OT OT OT OT WT WT WT WT WT OT WT WT WT APT APT APT APT APT APT APT APT APT YT APT Group I Group II Group III Group IV Fig.. HCA dendrogram of tea samples. ( GT samples, YT samples, WT samples, OT samples, BT samples, APT samples and RPT samples).

31 Figure Legends for Supporting Data Table S. The detail information of tea samples (types, brands, production places and contents of ten components). Table S. Linearity calibration curve factors, LOD and LOQ of the ten components. Table S. Repeatability, reproducibility and stability of the method. Table S. Recovery of the method Fig. S. Typical HPLC chromatograms of tea sample (GT) of (A) the combined extracts by the st + nd + rd times and (B) the extracts by the th time. Fig. S. The MS spectra and elemental composition of the ten components in positive ion mode.

32 Table S. The detail information of tea samples (types, brands, production places and contents of components). Type No. Brand name Production place Contents of ten components (mg/g, n=) Green tea () Yellow tea () GA TBM GC EGC C CAF EC EGCG GCG ECG GT Xihu Lungching Hangzhou, Zhejiang province GT Lungching Hangzhou, Zhejiang province GT Maofeng Emei, Sicuan province GT Biluochun Suzhou, Jiangsu province GT Yulu Enshi, Hubei province GT Green tea Anhui province GT Green tea Guangdong province GT Maofeng Fenggang, Guizou province GT Maojian Guizou province GT Zhuyeqing Emei, Sicuan province GT Queshe Guizou province GT Xihu Lungching Zhejiang province GT Xihu Lungching Zhejiang province GT Biluochun Jiangsu province GT Zhuyeqing Emei, Sicuan province GT Green tea Qianwei, Sicuan province GT Lungching Hangzhou, Zhejiang province YT Junshan Yinzhen Yueyang, Hunan province YT Junshan Yinzhen Yueyang, Hunan province YT Junshan Yinzhen Yueyang, Hunan province YT Yin Zhen Yueyang, Hunan province YT Huoshan Huangya Huoshan, Anhui province

33 White tea () Oolong tea () Aged pu-erh tea () YT Huangya Anhui province YT Huoshan Huangya Huoshan, Anhui province WT Baihao Yinzhen Fuding, Fujian province WT White tea Anji, Zhejiang province WT White tea Zhenan, Guizou province WT Mudanwang Fujian province WT Tianding Mudan Fujian province WT Shoumeiwang Fuding, Fujian province WT Shoumei Fujian province WT Baimudan Fuding, Fujian province OT Tieguanyin Anxi, Fujian province OT Malaocai Wuyi, Fujian province OT Tieguanyin Anxi, Fujian province OT Tieguanyin Anxi, Fujian province OT Tieguanyin Anxi, Fujian province OT Dahongpao Wuyi, Fujian province OT Dahongpao Wuyi, Fujian province OT Dahongpao Wuyi, Fujian province OT Tieguanyin Anxi, Fujian province OT Tieguanyin Anxi, Fujian province OT Oolong tea Taiwan OT Oolong tea Nantou, Taiwan OT Tieguanyin Anxi, Fujian province APT Qingpuer tea Puer, Yunnan province APT Puer tea Puer, Yunnan province APT Shengbing tea Puer, Yunnan province APT Shengbing tea Lincang, Yunnan province APT Qingbing tea Puer, Yunnan province

34 APT Qingpuer tea Puer, Yunnan province APT Puer tea Puer, Yunnan province APT Puer tea Puer, Yunnan province APT Qingpuer tea Kunming, Yunnan province APT Qingpuer tea Dali, Yunnan province RPT Shupuer tea Puer, Yunnan province RPT Shutuo tea Puer, Yunnan province RPT Shubing tea Lincang, Yunnan province RPT Shupuer tea Puer, Yunnan province Ripened RPT Shupuer tea Puer, Yunnan province pu-erh RPT Shubing tea Puer, Yunnan province tea () RPT Shubing tea Lincang, Yunnan province RPT Shupuer tea Puer, Yunnan province RPT Shuzhuan tea Kunming, Yunnan province RPT Shupuer tea Dali, Yunnan province RPT Shuzhuan tea Puer, Yunnan province BT Qimen black tea Qimen, Anhui province BT Lizhi black tea Yingde,Guangdong province BT Taicha Xinbei, Taiwan province Black tea BT Douji black tea Fengqing, Yunnan province () BT Tongmuguan Wuyi, Fujian province BT Jinjunmei Wuyi, Fujian province BT Black tea Lincang, Yunnan province BT Lizhi black tea Yingde, Guangdong province , not detected.

35 Table S. Linearity calibration curve factors, LOD and LOQ of the ten components. Peak No. RT (min) Component Range (mg /L) Equation R LOD (ng) LOQ (ng).0 GA 0.- y =. x TBM 0.- y =. x GC -0 y =.0 x EGC -0 y =. x C -0 y =. x CAF -0 y =. x EC -0 y =. x EGCG -0 y =.0 x GCG -0 y =. x ECG -0 y =. x

36 Table S. Repeatability, reproducibility and stability of the method. Component Repeatability RSD (%, n = ) Reproducibility (n = ) First day Third day Fifth day Interdays Calculated RSD Calculated RSD Calculated RSD RSD content (mg/g) (%) content (mg/g) (%) content (mg/g) (%) (%) Stability RSD (%, n = ) GA 0.. ± 0.0 a.. ± ± TBM 0.. ± ± ± GC 0.. ± ± ± EGC 0.. ± 0... ± 0... ± C 0.. ± ± ± CAF 0.. ± ± ± EC 0.. ± 0... ± 0... ± EGCG 0.. ± ± 0... ± GCG 0.. ± 0... ± 0... ± ECG 0.. ± ± 0... ± a The value is collected from the green tea sample of GT, and expressed as mean ± S.D. (n = ).

37 Table S. Recovery of the method. Component Added Added amount (mg) and recovery (%) at three spike levels 0% 0% 00% Recovery RSD Added Recovery RSD Added Recovery RSD Recovery Average RSD (mg) (%) (%) (mg) (%) (%) (mg) (%) (%) (%) (%) GA 0.0. ±. a. 0.. ±.... ±...0 ±.. TBM 0.0. ± ± ±... ±.0. GC 0.. ±.... ±.... ±... ±.. EGC. 0. ±.... ±.... ±... ±.. C.0. ±.0... ±..0.. ±... ±..0 CAF.0. ±...0. ± ±... ±.. EC.. ±.0... ±.... ±... ±.. EGCG.0. ±...0. ±...0. ±... ±..0 GCG.0. ±.... ±.... ±.. 0. ±.. ECG..0 ±.0... ±.... ±... ±.. a The value is collected from the green tea sample of GT, and expressed as mean ± S.D. (n = ).

38 x DAD - A:Sig=, Ref=0,0 T_++rd neg.d A st + nd + rd times x DAD - A:Sig=, Ref=0,0 T_th neg.d B th time < %, peak area 响应值与采集时间 ( 分钟 ) Fig. S. Typical HPLC chromatograms of tea sample (GT) of (A) the combined extracts by the st + nd + rd times and (B) the extracts by the th time. Notes: Brewing tea with hot water for a short while is the most popular way of tea drinking. Brewing period and brewing times were optimized, the results showed that that exhaustive extraction could be achieved when 0. g tea sample powder was extracted with 0 ml hot water at o C for min with intermittent shaking, three times (Figure S).

39 Components GA TBM MS spectra Elemental composition Formula Calculated Observed Error (m/z) (m/z) (mda) C H O C H O C H O C H O C H O C H O C H N O C H N O C H N O C H N C H N GC C H O C H O C H O EGC C H O C H O C H O C C H O C H O C H O C H O C H O

40 Components MS spectra Elemental composition Formula Calculated Observed Error (m/z) (m/z) (mda) C H N O C H N O CAF C H N EC C H O C H O C H O C H O C H O EGCG C H O C H O C H O GCG C H O C H O C H O ECG C H O C H O C H O Fig. S. The MS spectra and elemental composition of the ten components in positive ion mode.