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Naresuan University Journal 2012; 20(2)

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Optimization and Validation of the HPLC-Based Method for the Analysis of Gallic acid, Caffeine and 5 Catechins in Green Tea Theerapong Theppakorn and Sirirung Wongsakul Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand. Corresponding Author. E-mail address: [email protected] (T. Theppakorn) Received 20 October 2011; accepted 3 September 2012 Abstract A rapid and simple determination of gallic acid (G), caffeine (CF), (-)-epicatechin (EC), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-gallate (EGCG) and (+)-catechin (C) in green tea was developed and optimized. The optimized system consisted of a C18 reversed-phase column, an isocratic elution system (2.00 ml/min) of water:acetonitrile (87:13) containing 0.05% trifluoroacetic acid and diode array detector (210 nm). The tea catechins were identified by comparing absorption spectra and retention time with the reference standard under the identical conditions. The developed systems sufficiently separated G, EGC, C, EC, EGCG, CF and ECG within 7 min elution time at 30 Cํ . The validation of this method     showed that the detection limits of these compounds were 0.2 µg/ml with good linearity of up to 100 µg/ml. Assam green tea samples were analyzed using the developed systems and the levels of G, EGC, C, EC, EGCG, CF and ECG were reported. Keywords: analysis, catechins, caffeine, gallic acid, HPLC

Introduction Tea (Camellia sinensis L.), a widely consumed beverage in the world, comes in a variety of forms, scents and flavors. Green tea, oolong tea and black tea are made from the shoots of tea plants at different degrees of fermentation. Green tea, an unfermented tea, has been receiving much attention because it contains beneficial components, mainly polyphenols. Many biological functions of tea polyphenols have been discovered, including antioxidant activity (Martina, 2010; Kim et al., 2011), antimutagenic effects (Gupta et al., 2002; Wu et al., 2007), anti-obesity (Rains et al., 2011) and cardiovascular diseases prevention (Deka & Vita, 2011). Tea polyphenols exist in the biologically active group of the tea components, especially certain catechins, which make up as much as 30% (w/w) of the dry mass of tea. The major green tea catechins are (-)-epigallocatechin -3-gallate (EGCG), (-)-epigallocatechin (EGC), (-) -epicatechin-3-gallate (ECG) and (-)-epicatechin (EC). The level of green tea catechins varies according to varieties, climates, cultivation process and manufacturing process (Chen et al., 2010; Jayasekera et al., 2011; Wei et al., 2011). Moreover, the level of catechins depends mainly on the methods of extraction and analysis ( Labbé  et al., 2006; Yang et al., 2007; Komes et al., 2010). Therefore, it is important to establish a simple and reliable analytical

method for a routine determination of the levels of tea catechins. High performance liquid chromatography (HPLC) is normally used as the analytical technique to determine green tea catechins. The method has been optimized and validated as a simple and reliable analytical method (Khokhar et al., 1997; Bonoli et al., 2003; Wang et al., 2003). However, the timeconsuming nature of the use of HPLC to determine the compounds in green tea remains a major problem for routine analysis. In this paper we have developed a simple, fast and reliable isocratic HPLC method for simultaneous separation, identification and quantification of gallic acid, caffeine, (-)-epicatechin (EC), (-)-epicatechin-3-gallate (ECG), (-)epigallocatechin (EGC), (-)-epigallocatechin-3gallate (EGCG) and (+)-catechin (C) in green tea. Materials and Methods Standards, chemicals and samples Gallic acid (G), caffeine (CF), (-)-epigallocatechin (EGC), (+)-Catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin gallate (EGCG) and (-)epicatechin gallate (ECG) were purchased from SigmaAldrich (St. Louis, Missouri, USA). Acetonitrile (ACN), trifluoroacetic acid (TFA) and methanol (MeOH) (HPLC-grade) were purchased from Fluka (Buchs, Switzerland). Green tea samples were purchased from the tea shops in Chiang Rai province.

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Preparation of Standards The individual standard solutions of G, EGC, C, EC, EGCG, CF and ECG were prepared by dissolving them in a small volume of methanol, to generate a stock solution with a concentration of 1000 µg/ml.   The mixed standard stock solution was prepared by mixing an equal volume of each stock standard. Working standard solutions were prepared by the dilution of the mixed stock solution with water prior to HPLC analysis. Determination of dry matter content A dry matter content of tea was determined using a hot-air oven as the international standard method (ISO 1573, 1981). Sample extractions The methods used for sample extraction were based on international standard (ISO 14502-1, 2005). A brief procedure is as follows. First, a tea sample was ground and accurately weighed (0.200 ± 0.001 g) in a 10 ml extraction tube. A 5.0 ml of 70% (v/v) methanol was added and mixed on the vortex mixer. The extraction was performed in a water basin at 70 Cํ for 10 min, after which the extracted content was centrifuged at 3,500 rpm (3,000 g) for 10 min. This was followed by a repeated extraction, combining the extract to make up the volume to 10 ml with 70% (v/ v) methanol. The sample extract was filtered through a 0.45 µm PTFE filter prior to HPLC analysis. The sample was analyzed in duplicate. HPLC analysis HPLC analysis of standards and samples was conducted on Water 966 high performance liquid chromatography comprising a vacuum degasser, a quaternary pump, an auto-sampler, a thermostatted column compartment and a photo diode array detector. The column used was a Platinum EPS C18 reversed phase, 3 µm (53 mm x 7 mm) equipped with a guard column. The mobile phase eventually adopted for this study was water:acetonitrile (87:13) containing 0.05% (v/v) trifluoroacetic acid (TFA), with the flow rate of 2 ml/min. The absorption wavelength was selected at 210 nm. The column was operated at 30 Cํ . The sample injection volume was 20 µl . Peaks were identified by comparing their retention times and UV spectra in the range of 190-400 nm with standards, and by checking the purity of the peaks. Standard curves and limit of detection The standard curves were made using Microsoft Excel 2007. Triplicate injections were made, and peak areas were plotted against the concentrations to generate the standard curves using linear regressions. The limit

Naresuan University Journal 2012; 20(2)

of detection (LOD) was determined as the ratio of signal to noise that was equal to (or higher than) three (S/N = 3). The limit of quantification (LOQ) was determined as the ratio of signal to noise that was equal to (or higher than) ten (S/N = 10). Results and discussion Development of analytical methods Today’s laboratories are under greater demand to analyze more samples in less time. To help meet this demand, the smaller particles packed in a HPLC column has been made to decrease analysis time and increase HPLC throughput. The use of the smaller particles offers two main improvements to the chromatographic separation-increased resolution and speed. In this study, rapid analysis was a main goal. Therefore, a column with a smaller particle size (3µm,  7 mm ID x 53 mm) was selected. We examined the optimal conditions for the analysis of the five catechins (EGC, C, EC, EGCG and ECG), gallic acid (G) and caffeine (CF) in green tea samples using a simple isocratic system for HPLC. In most studies, water/methanol/acid, water/ acetonitrile/ethyl acetate and water/acetonitrile/acid methods have been used for catechin analysis (Yayabe et al., 1989; Goto et al., 1996; Wang et al., 2000; Nishitani & Sagesaka, 2004). In this study, three mobile phases widely used for tea analysis were tested. These are water/acetonitrile (87:13), water/ acetonitrile/ethyl acetate (86:12:2) and water/ methanol (87:13). It was found that the water/ methanol (87:13) method provided poor resolution and low chromatographic efficiency (Figure 2). The use of water/acetonitrile (87:13) and water/acetonitrile/ ethyl acetate (86:12:2) resulted in good separation. The optimal mobile phase is water/acetonitrile (87:13) due to the short running time consumed. Next, we studied the effect of acid on the chromatographic efficiency. It has been reported that the presence of acid is essential to complete separation (Dalluge et al., 1998). However, we found that 0.05% TFA did not benefit the separation of catechins but improved the peak sharpness (Figure 3). The elution order was G, EGC, C, EC, EGCG, CF and ECG, respectively. Next, the compositions of the mobile phase, namely, water/acetonitrile containing 0.05% TFA, were varied (water:acetonitrile = 90:10, 87:13, 85:15 and 80:20). Figure 4 shows that the increase in acetonitrile resulted in short elution time. However, a peak overlap was found at 15% and 20% acetonitrile

Naresuan University Journal 2012; 20(2)

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(Figure 4 C and D). The use of 13% acetonitrile is possibly the optimal composition due to good resolution and short time. The flow rate of the mobile phase (water/acetonitrile (87:13) containing 0.05% TFA) was further optimized (Figure 5). We found the peak overlap at the flow rate of 2.5 ml/min (peaks of EGC/ C and EC/EGCG). Good separation was achieved when the flow rates of 1.0, 1.5 and 2.0 ml/min were used. For good separation and rapid analysis, the flow rate of 2.0 ml/min was selected. At this flow rate, all components could be eluted within 7 min (Figure 5C), faster than the elution time previously reported by Horie & Kohata (2000), Wang et al. (2000), Zuo et al. (2002), Nishitani & Sagesaka (2004) and Sharma et al. (2005). Column temperatures play an important role in the separation of compounds by HPLC. We subsequently

investigated the effect of temperatures on chromatographic efficiency. It is clear, as Figure 6 shows, that the increase in temperatures (from 30 to 45 Cํ ) resulted in shorter elution times; however, peak overlaps occurred (Figure 6 B, C and D). The optimal temperature was at 30 Cํ . Wavelengths have been used for detection of catechins and caffeine in green tea, varying from 210 to 280 nm. (Goto et al., 1996; Dalluge et al., 1998; Wang et al., 2000; Horie & Kohata, 2000). Figure 7 shows the chromatograms of standard solution at 210, 254 and 280 nm. The absorbance of standards at 210 nm was obviously greater than those at 280 and 254 nm, respectively. Thus we selected 210 nm as the appropriate wavelength for detection of catechins, caffeine and garlic acid.

OH

OH OH

HO

OH

O

HO

O

OH

OH

OH

OH

OH

(-)-Epicatechin (EC)

(-)-Epigallocatechin (EGC) OH

OH OH

HO

OH

O

HO

O

OH

O

O OH

OH

OH

OH

O

O OH

OH

OH

OH

(-)-Epicatechin gallate (ECG)

(-)-Epigallocatechin gallate (EGCG)

OH

O

HO

OH

H 3C

O HO

HO

O

C

N N

OH

N O

N

HO OH OH

CH 3

Gallic acid

(+)-Catechin (C)

Figure 1 Structure of 5 catechins, gallic acid and caffeine.

CH 3

Caffeine

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Naresuan University Journal 2012; 20(2) A 



C

Figure 2 Effects of mobile phases on the separation of standard substance mixtures (100 mg/ml). The analysis was performed on Platinum EPS C18, 100 Å , 3 µm, 53 mm x 7mm, flow rate 2 ml/min, temperature 30 Cํ , detection 254 nm, mobile phase (A) H2O:ACN, 87:13 v/v, (B) H2O:ACN:EtOAC, 86:12:2 v/v and (C) H2O: MeOH, 87:13 v/v.

Naresuan University Journal 2012; 20(2)

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Figure 3 Effects of added acid on the separation of standard substance mixtures (100 mg /ml). The analysis was performed on Platinum EPS C18, 100 Å , 3 µm , 53 mm x 7 mm, flow rate 2 ml/min, temperature30 ํC, detection 210 nm, mobile phase H2O:ACN (87:13) in the presence (A) and absence (B) of trifluoroacetic acid.







D

Figure 4 Effects of mobile phase compositions on the separation of standard substance mixtures. The analysis was performed on Platinum EPS C18, 100 Å , 3 µm , 53 mm x 7mm, flow rate 2 ml/min, temperature 30 ํC, detection 210 nm, mobile phase H2O:ACN (A) 90:10, (B) 87:13, (C) 85:15 and (D) 80:20.

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Naresuan University Journal 2012; 20(2)

Figure 5 Effects of flow rate of mobile phase on the separation of standard substance mixtures. The analysis was performed on Platinum EPS C18, 100 Å , 3 µm , 53 mm x 7mm, mobile phase water:acetonitrile 87:13% containing 0.05% TFA, temperature 30 ํC, detection 210 nm, and flow rate of (A) 1.0, (B) 1.5, (C) 2.0 and (D) 2.5 ml/min.

Figure 6 Effects of temperatures on the separation of standard substance mixtures. The analysis was performed on Platinum EPS C18, 100 Å , 3µm , 53 mm x 7mm, mobile phase water:acetonitrile 87:13% containing 0.05% TFA, flow rate 2.0 ml/ min, detection 210 nm, at temperatures (A) 30, (B) 35, (C) 40 and (D) 45 ํC.

Validation of analytical methods Calibration graphs for the catechins, caffeine and gallic acid were constructed using Microsoft Excel 2007 (Figure 8). All the analytes exhibited good linearity over the range tested, with a correlation coefficient (r) between 0.9958 and 0.9999 (Table 1). The linearity of all compounds was in the range of 1-100 mg/ml. The LOD and LOQ of all analytes were 0.2 and 1.0 mg/ml respectively. Green tea was extracted and analyzed for gallic acid, caffeine and 5 catechins

using the developed method. A typical chromatogram of the green tea is shown in Figure 9. Precision and accuracy were evaluated using recovery tests (n = 7), adding known amounts of the standard solution (1, 20, 60 and 80 mg/ml) to a green tea extract. The recovery rates of all of the compounds were in the range of 84.7103.7%. Relative standard deviations (%RSD) of all of the compounds were less than 10%. To check the specificity, we performed peak purity diode-array analysis and confirmed that each chromatographic peak

Naresuan University Journal 2012; 20(2)

was attributable to a single component (data not shown). All these results suggested that the developed method was reliable. Analysis of green tea samples As the rapidly optimized method has been found applicable with acceptable accuracy and precision, this method was used for determining gallic acid, caffeine and 5 catechins in 6 Assam green tea samples produced from Camellia sinensis var. assamica. Table 2 shows that the average gallic acid was 1.23 %w/w dry basis. The caffeine content of six green tea samples ranged from 3.27% to 3.71% w/w, with a mean of 3.44%w/ w. This was quite similar to the caffeine content in

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earlier reports, 2.94-3.62% (Nishitani & Sagesaka, 2004) and 2.29-3.82% (Wang et al., 2000). Table 2 indicates that all Assam green tea samples contain EGC, C, EC, EGCG and ECG. The major catechins were ECG, EGCG and EC, with a concentration of 2.94, 2.62 and 2.23%w/w, respectively. It is quite impractical to compare the individual catechins with other published works because many factors such as species, cultivating season, plucking standards or methods, horticultural conditions and processing can all influence the catechin contents (Chen et al., 2010; Jayasekera et al., 2011; Wei et al., 2011).





C

Figure 7 The chromatogram of standard substance mixtures. The analysis was performed on Platinum EPS C18, 100 Å , 3 µm , 53 mm x 7 mm, mobile phase water:acetonitrile 87:13% containing 0.05% TFA, flow rate 2.0 ml/min, temperature 30 ํC and detection wavelength at (A) 210, (B) 254, and (C) 280 nm.

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Naresuan University Journal 2012; 20(2)

Figure 8 Calibration curves for gallic acid (G), caffeine (CF), (-)-epigallocatechin (EGC), (+)-Catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG).

Naresuan University Journal 2012; 20(2)

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Figure 9 A typical HPLC chromatogram of Assam green tea. The analysis was performed on Platinum EPS C18, 100 Å , 3 µm, 53 mm x 7 mm, mobile phase water:acetonitrile 87:13% containing 0.05% TFA, flow rate 2.0 ml/min, temperature 30 ํC and detection wavelength at 210 nm. Table 1 Results of validation of the analytical method (n = 7) G

CF

EGC

Compounds C

1-100 0.2 1.0 0.9958 93.4±7.9 98.4±8.0 97.8±4.4 98.6±4.3 8.5 8.1 4.5 4.4

1-100 0.2 1.0 0.9999 83.1±4.9 95.4±4.4 94.6±4.3 99.9±1.1 5.9 4.6 4.5 1.1

1-100 0.2 1.0 0.9998 84.7±8.2 93.9±8.6 93.8±7.8 94.7±4.3 9.7 9.2 8.4 4.6

1-100 0.2 1.0 0.9998 96.9±8.5 96.1±6.9 94.8±4.5 94.9±4.2 8.8 7.2 4.8 4.4

Parameters Linearity LOD LOQ r

%Recovery

%RSD

(µg/ml) (µg/ml) (µg/ml) 1.0 µg/ml 20 µg/ml 60 µg/ml 80 µg/ml 1.0 µg/ml 20 µg/ml 60 µg/ml 80 µg/ml

EC

EGCG

ECG

1-100 0.2 1.0 0.9999 95.9±8.9 89.8±8.1 94.1±4.2 93.9±3.9 9.3 8.9 4.5 4.1

1-100 0.2 1.0 0.9994 93.6±8.3 103.8±3.8 96.2±5.3 95.4±3.2 8.8 3.7 5.5 4.3

1-100 0.2 1.0 9.9996 90.0±8.0 94.1±4.3 94.4±4.1 97.7±1.2 8.9 4.5 4.3 1.2

Table 2 Contents of gallic acid, caffeine and 5 catechins in Assam green tea samples Assam green tea 1 2 3 4 5 6 Min Max Mean

G 1.41 1.31 1.65 1.11 1.03 0.89 0.89 1.65 1.23

CF 3.71 3.43 3.44 3.35 3.27 3.45 3.27 3.71 3.44

Content (%w/w dry basis) EGC C EC 1.60 1.70 2.88 1.95 1.51 2.29 1.70 2.15 2.62 2.16 1.41 1.31 2.48 1.61 2.39 0.70 1.53 1.87 0.70 1.41 1.31 2.48 2.15 2.88 1.77 1.65 2.23

EGCG 2.20 2.74 2.44 3.69 3.47 1.16 1.16 3.69 2.62

ECG 3.53 2.90 3.45 1.65 2.84 3.24 1.65 3.53 2.94

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Naresuan University Journal 2012; 20(2)

Conclusion The developed isocratic HPLC method allows rapid and simultaneous analysis of gallic acid (G), caffeine (CF), (-)-epicatechin (EC), (-)-epicatechin-3gallate (ECG), (-)-epigallocatechin (EGC), (-)epigallocatechin-3-gallate (EGCG) and (+)-catechin (C) in green tea within 7 min. This method is simple, rapid and can be used for fast routine analysis. Acknowledgements The author thanks Mae Fah Luang University for funding of this research. References Bonoli, M., Pelillo, M., Toschi, T. G. & Lercker, G. (2003). Analysis of green tea catechins: comparative study between HPLC and HPCE. Food Chemistry, 81, 631-638. Chen, Y., Jiang, Y., Duan, J., Shi, J., Xue, S., & Kakuda, Y. (2010). Variation in catechin contents in relation to quality of ‘Huang Zhi Xiang’ Oolong tea (Camellia sinensis) at various growing altitudes and seasons. Food Chemistry, 119, 648-652.

ISO 1502-1. (2005). Determination of substances characteristic of green and black tea-Part 1: Content of total polyphenols in tea-Colorimetric methods using Folin-Ciocateu reagent. Switzerland: International Standard Organization. ISO 1573. (1981). Determination of loss in mass at 103 ํ C. Methods of test for tea. Switzerland: International Standard Organization. Jayasekera, S., Molan, A. L., Garg, M. & Moughan, P. J. (2011). Variation in antioxidant potential and total polyphenol content of fresh and fully-fermented Sri Lankan tea. Food Chemistry, 125, 536-541. Khokhar, S., Venema, D., Hollman, P. C. H., Dekker, M. & Jongen, W. (1997). A RP-HPLC method for the determination of tea catechins. Cancer Letters, 114, 171-172. Kim, Y., Goodner, K. L., Park, J.-D., Choi, J. & Talcott, S. T. (2011). Changes in antioxidant phytochemicals and volatile composition of Camellia sinensis by oxidation during tea fermentation. Food Chemistry, 129, 1331-1342.

Komes, D., Horžić, D., Belščak, A., Ganić, K. K., & Dalluge, J. J., Nelson, B. C. Thomas, J. B. & Sander, Vulić, I. (2010). Green tea preparation and its influence L. C. (1998). Selection of column and gradient elution on the content of bioactive compounds. Food Research system for the separation of catechins in green tea using International, 43, 167-176. high performance liquid chromatography. Journal of Chromatography A, 793, 265-274. Labbé, D., Tremblay, A. & Bazinet, L. (2006). Effect of brewing temperature and duration on green tea Deka, A. & Vita, J.A. (2011). Tea and cardiovascular catechin solubilization: Basis for production of EGC disease. Pharmacological Research, 64, 136-145. and EGCG-enriched fractions. Separation and Purification Technology, 49, 1-9. Goto, T., Yoshida, Y., Kiso, M. & Nagashima, H. (1996). Simultaneous analysis of individual catechins Martina, B. (2010). Comparison of the antioxidant and caffeine in green tea. Journal of Chromatography capacity and the antimicrobial activity of black and A, 749, 295-299. green tea. Food Research International, 43, 1379-1382. Gupta, S., Saha, B. & Giri, A. K. (2002). Comparative antimutagenic and anticlastogenic effects of green tea and black tea: a review. Mutation Research/Reviews in Mutation Research, 512, 37-65.

Nishitani, E. & Sagesaka, M. Y. (2004). Simultaneous determination of catechins, caffeine and other phenolic compounds in tea using new HPLC method. Journal of Food Composition and Analysis, 17, 675-685.

Hories, H. & Kohata, K. (2000). Analysis of tea components by HPLC and HPCE. Journal of Chromatography A, 881, 425-438.

Rains, T. M., Agarwal, S. & Maki, K.C. (2011). Antiobesity effects of green tea catechins: a mechanistic review. The Journal of Nutritional Biochemistry, 22, 1-7.

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Sharma, V., Gulati, A., Ravindranath, S. D., & Kumar, V. (2005). A simple and convenient method for analysis of tea biochemicals by reverse phase HPLC. Journal of Food Composition and Analysis, 18, 583594. Wang, H., Helliwell, K. & You, X. (2000). Isocratic elution system for determination of catechins, caffeine and gallic acid in green tea using HPLC. Food Chemistry, 68, 115-121. Wang, H., Provan, G. J. & Helliwell, K. (2003). HPLC determination of catechins in tea leaves and tea extracts using relative response factors. Food Chemistry, 81, 307-312. Wei, K., Wang, L., Zhou, J., He, W., Zeng, J., Jiang, Y. & Cheng, H. (2011). Catechin contents in tea (Camellia sinensis) as affected by cultivar and environment and their relation to chlorophyll contents. Food Chemistry, 125, 44-48. Wu, S.-C., Yen, G.-C., Wang, B.-S., Chiu, C.-K., Yen, W.-J., Chang, L.-W. & Duh, P.-D. (2007). Antimutagenic and antimicrobial activities of pu-erh tea. LWT - Food Science and Technology, 40, 506512. Yang, D.-J., Hwang, L. S. & Lin, J.-T. (2007). Effects of different steeping methods and storage on caffeine, catechins and gallic acid in bag tea infusions. Journal of Chromatography A, 1156, 312-320. Yayabe, F., Kinugasa, H. & Taeko, T. (1989). A simple preparative chromatographic separation of green tea catechins. Nippon Nogeikagaku Kaishi, 63, 845847. Zuo, Y., Chen, H. & Deng, Y. (2002). Simultaneous determination of catechins, caffeine and gallic acids in green, Oolong, black and pu-erh teas using HPLC with a photodiode array detector. Talanta, 57, 307-316.

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