Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection

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1 Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection Mitsuaki Sano,* a Michiko Tabata, a Masazumi Suzuki, a Masakuni Degawa, a Toshio Miyase a and Mari Maeda-Yamamoto b THE ANALYST FULL PAPER a School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka , Japan. sano@ys2.u-shizuoka-ken.ac.jp b National Research Institute of Vegetables, Ornamental Plants and Tea, Ministry of Agriculture, Forestry and Fisheries, 2769 Kanaya, Shizuoka , Japan Received 19th March 2001, Accepted 18th April 2001 First published as an Advance Article on the web 23rd May 2001 A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of twelve tea catechins including four major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG); four of their epimers at the C-2 position, C, GC, CG and GCG; and four methylated catechin derivatives, epigallocatechin-3-o-(3-o-methyl)gallate, gallocatechin-3-o-(3-o-methyl)gallate, epigallocatechin-3-o-(4-o-methyl)gallate and epicatechin-3-o-(3-o-methyl)gallate. These catechins were separated on an ODS C 18 reversed-phase column by isocratic elution with 0.1 M NaH 2 PO 4 buffer (ph 2.5) acetonitrile (87+13) containing 0.1 mm EDTA 2Na. The detection limits (S/N = 3) of these catechins were approximately pmol ml 21 at an applied voltage of 600 mv. Extracting these catechins from tea leaf powder with H 2 O acetonitrile (1+1) at 30 C for 40 min inhibited the epimerization at C-2 significantly from these epicatechins compared to extraction with hot water at 90 C. This analytical method is sensitive to and appropriate for the simultaneous determination of various biologically active catechins in green tea. Introduction The major catechins in green tea leaves (Camellia sinensis L.) are (2)-epigallocatechin gallate (EGCG), (2)-epigallocatechin (EGC), (2)-epicatechin gallate (ECG) and (2)-epicatechin (EC), which normally comprise more than 75% of tea polyphenols. Various biological and pharmacological properties of tea catechins have been reported. In particular, EGCG is known to exhibit more potent beneficial effects, including antioxidative, 1,2 anti-mutagenic, 3 anti-mutagenic/anti-carcinogenic, 4 6 anti-allergic 7,8 and anti-atherosclerotic properties, 9,10 than the other catechins. Gallocatechin gallate (GCG) which is an epimer of EGCG at the C-2 position, is often detected in tea infusions. GCG also has a potent bioavailability in some bioassays. 11,12 Recently, we demonstrated that two O-methylated derivatives of EGCG exhibit strong anti-allergic properties on type I and type IV allergies. 13,14 The methylated catechins are concentrated in limited tea cultivars, and the concentrations are much lower than those of the major catechins. Several analytical methods using high-performance liquid chromatography (HPLC) combined with UV detection, 15,16 electrochemical detection (ECD) or chemiluminescence detection 20 have been reported to determine major catechins in tea leaves and biological samples. We previously reported an HPLC method with ECD for the determination of four epicatechins in tea infusion 17 and blood plasma. 21 The HPLC method with ECD is sensitive to and selective for the determination of polyphenols, especially trace amounts of catechins such as methylated catechins and epimerization products. There are few reports on the simultaneous determination of the catechins including methylated catechins detected in tea infusions. In this study, we developed a simultaneous determination method for twelve tea catechins (Fig. 1) including four major catechins, four of their epimers and four methylated catechin derivatives by HPLC with ECD. We also describe that extraction with H 2 O acetonitrile at 30 C prevents epimerization of epicatechins. Experimental Reagents and samples Tong ting oolong tea was obtained from markets in Japan and Taiwan. All other teas were cultivated at the plantation of the National Research Institute of Vegetables, Ornamental Plants and Tea in Kanaya, Shizuoka, Japan. Freshly picked tea leaves were dried in a microwave oven and stored in a refrigerator before analysis and isolation of catechins. All tea catechins except EGCG4 Me were isolated from the cultivar Benihomare. EGCG4 Me was isolated from Tong ting oolong tea. The extraction and isolation of methylated catechins was performed according to the method previously reported. 13 Their purities were confirmed by 1 H-NMR. Some catechins (EGCG, EC, ECG, EGC) purchased from Kurita Co. (Tokyo, Japan) were also used. Propyl gallate as the internal standard (I.S.), EDTA 2Na and acetonitrile were purchased from Wako Pure Chemical Co. Ltd. (Osaka, Japan). Water used was purified in a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals were of reagent grade. Apparatus The HPLC system consisted of a pump (Shimadzu LC-10AD, Kyoto Japan), an automated sample injector (Shimadzu SIL- 10A), a column oven (Shimadzu, CTO-10AC), a system 816 Analyst, 2001, 126, DOI: /b102541b This journal is The Royal Society of Chemistry 2001

2 controller (Shimadzu, SCL-10A) and an ECD (Shiseido, Nanospace SL-1, Tokyo, Japan). The recording and integrating device was a Chromatopac (Shimadzu, CR 7A plus). The separation column was a TSK gel ODS 80Ts reversed-phase column ( mm id, Tosoh, Tokyo, Japan). Preparation of tea samples Standard catechins dissolved in acetonitrile distilled water (1+1) were used to construct calibration plots for the determination of catechin concentrations in tea infusion. Tea leaves were pulverized into powder for analysis. Extraction of catechin from tea leaves was done by a slight modification of the procedure of Suematsu et al. 22 Catechins were extracted from 50 mg of the tea leaf powder by 10 ml acetonitrile distilled water (1+1) at 30 C for 40 min in a water bath with shaking (120 cycles min 21 ). The extracts were centrifuged at 3000 rpm at 4 C for 10 min, and the supernatant was diluted 20-fold with 50% aqueous acetonitrile containing internal standard. Twenty ml of the sample filtered through a membrane filter (pore size 0.45 mm: type Ekikurodisk 13, Gelman Sci. Japan, Tokyo, Japan) was injected into the HPLC apparatus. Each catechin in the tea infusion was identified by the retention time relative to standards. Epimerization Tea infusions were obtained by extraction from green tea leaf powder with distilled water at 30 C for 40 min. The extracts were passed through a 0.45 mm membrane filter to remove tea solids. An aliquot of the filtrate was maintained in a water bath at 90 C or 30 C for 30 min for the examination of C-2 epimerization of epicatechins. catechins were detected electrochemically at an applied potential of 600 mv versus Ag/AgCl. Results and discussion HPLC separation and detection of various catechins The twelve varieties of standard catechins (5 mm) and the internal standard were sufficiently separated by use of an ODS 80Ts reversed-phase column under the conditions described in the Analytical method section (Fig. 2). The retention times obtained were 5.5, 7.4, 10.0, 15.4, 17.9, 22.4, 32.9, 35.3, 50.1, 57.4, 62.7, 71.0 and min for GC, EGC, C, EC, EGCG, GCG, EGCG4 Me, EGCG3 Me, ECG, CG, GCG3 Me, n- propyl gallate (I.S.) and ECG3 Me, respectively. The detection limits (S/N = 3) of these catechins were approximately 10 pmol ml 21 (EGCG, EGC) 40 pmol ml 21 (ECG3 Me) at the applied voltage of 600 mv. Fig. 3 and Fig. 4 show typical HPLC chromatograms of the extracts from Tong ting oolong tea and Benihomare green tea. Tong ting oolong tea contains two O- methylated EGCG (EGCG3 Me and EGCG4 Me). Ascorbic acid, which exists at concentrations of mg per 100 g in tea leaves, is detected by ECD. It is, however, not trapped on the column. Purine compounds such as caffeine are not detected under these conditions. The small, broad peak that appeared at min in the chromatogram (see Fig. 4) was at a retention time that agreed with the standard ECG3 Me shown in Fig. 2. Analytical method The optimal mobile phase for the separation of tea catechins was 0.1 M NaH 2 PO 4 buffer (ph 2.5) containing 0.1 mm EDTA 2Na acetonitrile (87+13) at a flow rate of 1.0 ml min 21. The HPLC column was maintained at 30 C in an oven. The Fig. 2 HPLC chromatogram of standard catechins. A mixture of catechins (5 mm) and n-propyl gallate as internal standard (I.S.) was used for the HPLC-ECD analysis. Peaks are 1: GC; 2: EGC; 3: C; 4: EC, 5; EGCG; 6: GCG; 7: EGCG4 Me; 8: EGCG3 Me; 9: ECG; 10: CG; 11: GCG3 Me; 12: I.S. and 13: ECG3 Me. Fig. 1 Chemical structures of tea catechins and O-methylated derivatives of EGCG. Analyst, 2001, 126,

3 However, the components in the fraction eluted at that retention time were identified as a mixture of ECG3 Me and epiafzelechin-3-o-gallate (C 22 H 18 O 9 ), 23 one of the tea polyphenols, by 1H-NMR and FAB-MS. The elution of the two polyphenols, therefore, overlapped, and separation was impossible under these HPLC conditions. Further experimentation with a solvent gradient system after 90 min retention time is needed to separate these two tea components. Recovery of catechins from tea leaves Standard catechins (EGCG, ECG, EGC, EC, GCG, C, CG, GC, EGCG3 Me, EGCG4 Me) were added to the tea powder at concentrations of 1% or 5% of the weight of dry tea leaf prior to extraction. The catechins were extracted from the tea according to the extraction procedure described in the Experimental section. Table 1 lists the recovery (%) of each added catechin. Overall recoveries of catechins added to tea leaf powder at a concentration of 1% or 5% of weight tea powder were from 84% to 99%. Catechin concentrations of tea leaves Catechins in green tea prepared from some tea cultivars and Tong ting oolong tea were examined by the proposed HPLC method (Table 2). The EGCG3 Me content in the cultivars Benifuuki, Benifuji and Benihomare, which are classified as Assam hybrids, was higher than the other tea cultivars. The EGCG4 Me was localized exclusively in Tong ting oolong tea. Epimerization in extraction procedure Some epimeric isomers of epicatechins were detected in aqueous tea infusions, in particular, in infusions extracted with Fig. 3 Representative chromatogram of tea infusion. Catechins of Tong ting oolong tea leaf powder were extracted with 50% aqueous acetonitrile at 30 C for 40 min. Peaks are 1: GC; 2: EGC; 3: C; 4: EC; 5: EGCG; 6: GCG; 7: EGCG4 Me; 8: EGCG3 Me; 9: ECG and 12: I.S. Fig. 4 Chromatograms of tea infusions extracted for 40 min with (A) 50% aqueous acetonitrile at 30 C and (B) hot water at 90 C. Tea leaf powder used was prepared from cultivar Benihomare green tea. Peaks are 1: GC; 2: EGC; 3: C; 4: EC; 5: EGCG; 6: GCG; 8: EGCG3 Me; 9: ECG; 10: CG; 11: GCG3 Me; 12: I.S. and 13: ECG3 Me and epiafzelechin-3-o-gallate. 818 Analyst, 2001, 126,

4 Table 1 Recovery of catechins from green tea leaf powder Recovery (%) b Added (%) a EGCG EGC ECG EC EGCG3 Me EGCG4 Me GCG GC CG C ± ± ± ± ± ± ± ± ± ± c 87.6 ± ± ± ± 3.0 a Each standard catechin was added to the tea (cultivar Benihomare) powder at a concentration of 1% or 5% of weight of dry tea leaf. b Values are mean ± s for triplicate determinations. c The recovery tests at the concentration of 5% were carried out only for major epicatechins (EGCG, EGC, ECG, EC). Table 2 Catechin concentrations in various tea leaves a Dry wt. of tea leaves (%) Tea variety EGCG EGC ECG EC EGCG3 Me EGCG4 Me GCG GC CG C Green tea Inzatsu Okuyutaka Gokou Sayamamidori Seishin oolong Trace Benifuuki Benifuji Benihomare Yabukita Yutakamidori Tong ting oolong tea A B C Trace 0.08 D Trace 0.03 a Values are means of duplicate determinations. The sample preparation method was described in the Experimental section. hot water. Since catechin has two asymmetric carbon atoms (C- 2 and C-3) in a C ring, four epimerization products are possible in theory. However, epimers of epicatechins with the carbon at the C-2 position were exclusively detected in tea infusion. We examined C-2 epimerization in extractions with hot water or with 50% aqueous acetonitrile, which is known to be a good extractant of major tea catechins. 22 Fig. 4 shows the chromatograms of catechins extracted from the cultivar Benihomare with (A) 50% aqueous acetonitrile at 30 C and (B) hot water at 90 C for 40 min. The cultivar Benihomare contains EGCG3 Me but not EGCG4 Me (see Table 2). The concentrations of the epimers corresponding to the epicatechins were much higher in hot water infusions than in the acetonitrile infusions. The percent values of GC/EGC, C/EC, GCG/EGCG, CG/ECG and GCG3 Me/EGCG3 Me in hot water infusions were 59.49%, 28.73%, 57.58%, 25.32% and 38.10%, respectively, and the values in the acetonitrile infusion were 1.65%, 7.43%, 5.21%, 1.57% and 3.48%, respectively. The epimerization in extracts obtained with hot water at 90 C progressed time-dependently (data not shown). Fig. 5 shows the epimerization of catechins in tea infusion prepared as described in the Experimental section. Incubation of the infusion at 30 C for 30 min did not cause epimerization of epicatechins, while, incubation at 90 C for 30 min resulted in marked epimerization. The results suggest that most of the epimers of epicatechins detected in tea leaves might be produced by steam treatment during the process of green tea production. Recently, GCG has been reported to exhibit a higher inhibitory effect than EGCG against activator protein 1 activity 12 or type I allergy. 11 Therefore, if the epimer has a high bioavailability, the available epimer can be easily produced from tea leaves by extractive processes using heat treatment. The proposed HPLC method with ECD is thus sensitive and useful for the detection of various catechins in tea leaves. This Fig. 5 Conversion of epicatechins to the corresponding epimers in tea infusion by heat treatment. Tea infusion without tea leaf residues was prepared as described in the Experimental section. The infusion was incubated for 30 min at 30 C or 90 C. method could also be applicable to studies on the epimerization or metabolic fate of tea catechins. Acknowledgement This work was supported by a grant from the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). References 1 N. Salah, N. J. Miller, G. Paganga, L. Tijburg, G. P. Bolwell and C. Rice-Evans, Arch. Biochem. Biophys., 1995, 322, 339. Analyst, 2001, 126,

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