You are on page 1of 6

Food Chemistry 81 (2003) 307–312 www.elsevier.


HPLC determination of catechins in tea leaves and tea extracts using relative response factors
Huafu Wang*, Gordon J. Provan, Keith Helliwell
R & D Department, William Ransom & Son plc, Hitchin, Herts SG5 1LY, UK Received 19 February 2002; received in revised form 2 October 2002; accepted 2 October 2002

Abstract A simple high performance liquid chromatographic analysis of tea catechins using relative response factors has been developed. The separation system consisted of a C18 reversed-phase column, a gradient elution system of methanol/water and orthophosphoric acid, and a photodiode array detector. Relative response factors for catechins are given on different columns and relative to different references. It has been shown that the relative response factors for catechins are quite similar at 210 nm of detection under different analytical conditions (different columns, different elution systems, and different HPLC instruments). (+)-Catechin was selected as the reference compound for calculating the relative response factors of the catechins. Using this method, not every catechin is needed as a reference standard, making the method ideal for rapid, routine analysis, especially for those laboratories where catechin standards are not readily available. The method is applicable to all kinds of tea, tea extracts and some tea containing products. It is especially useful for the determination of (+)-gallocatechin and (+)-catechin, which often are regarded as being present below detectable limits when detected at 280 nm, and (À)-catechin gallate, which takes a long time to elute in isocratic systems. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Tea; Catechins; Relative response factors; HPLC

1. Introduction Tea is the most popular beverage after water throughout the world. Tea was first used in China for its medicinal properties 5000 years ago (Balentine, 1997; Cheng & Chen, 1994), but it is only in the last couple of decades that the potential health benefits of this ancient beverage have been documented on a scientific basis. A growing body of evidence from both human and animal studies suggests that regular consumption of green tea can reduce the incidence of a variety of cancers, including colon, pancreatic, and stomach cancers, and other diseases (Alexis, Jones, & Stiller, 1999; Balentine, Wiseman, & Bouwens, 1997; Dreostic, Wargovich, & Yang, 1997; Steiawan et al., 2001; Wang, Provan, & Helliwell, 2000). It is generally believed that catechins, the principle

* Corresponding author. Tel.: +44-1462-437615; fax: +44-1462420528. E-mail address: (H. Wang).

bioactive compounds in tea, are responsible for the claimed therapeutic activity of tea. In addition to the direct consumption of tea either by brewing loose leaves or tea bags or in a ready-to-drink form, in recent years, there have been more and more applications for tea extracts especially in the nutraceutical, and food areas. Therefore it is essential to be able to offer consumers a consistent level of catechins in their products. Tea leaves, their extracts and the consumer products themselves need to be standardised and routinely assayed. Furthermore, any potential health implications require data generation on the content of catechins in tea leaves, their extracts and products. For these purposes, much attention has been paid to developing analytical methods for tea catechins (Bronner & Beecher, 1998; Dalluge & Nelson, 2000; Dalluge, Nelson, Thomas, & Sander, 1998; Goto, Yoshida, Kiso, & Nagashima, 1996; Khokhar, Venema, Hollman, Dekker, & Jongen, 1997; Wang, Helliwell, & You, 2000). Analysis of catechins is generally accomplished by HPLC using a reversed stationary phase and various

0308-8146/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0308-8146(02)00510-1

Materials and methods 2. The column was maintained at 30  C. Chinese Academy of Agricultural Sciences. The water used in HPLC and sampling was prepared with a Super Purity Water System (Purite Ltd. Mobile phases consisted of 0. RRF: the average relative response factor of that catechin to the reference catechin. caffeine and theobromine were purchased from Sigma Chemical Co (Dorset. 2. 5– 7 min. linear gradient from 20 to 24% B. thermostatted column compartment. The relative response factors were calculated as the ratio of the response factor for each catechin to that of the chosen reference catechin. 20% B.1. the high cost and instability of some catechin reference standards have limited their application. Analytical determinations An HP 1100 series liquid chromatograph system comprising vacuum degasser. Vsamp: volume of sample solution (ml).2. England) with a resistivity over 17. quaternary pump. The sample injection volume was 10 ml. 2. The extraction solution was filtered into a 50 ml volumetric flask. UK) with a Kingsorb 5 mm C18 (30  4. Elution was performed at a solvent flow rate of 1 ml/min. 20–25 min. Calculation of relative response factors and quantification of catechins The standard solution was analysed under the conditions detailed above. (À)-catechin gallate [(À)-CG] and gallic acid.000 rpm for 10 min prior to HPLC analysis. (+)-catechin [(+)-C]. Approximately 1 ml of the sample solution was centrifuged at 13. v/v). By using these relative response factors catechin analysis can be carried out with only one selected catechin as a reference standard. Wsamp: sample weight (mg). The gradient was as follows: 0–5 min. UK). v/v) by sonication for 20 min. Tea dry extract was either produced from green tea by the reporting company or purchased from other manufactures. Roasted green tea (RGT) and Keemun black tea were obtained from the Tea Research Institute.6 mm) (phenomenex1. The elution system employs simple mobile phases containing methanol and water. UK). Rfc: response factor of the catechin standard [(mg/ml)/mAU*s]. UK. Working standard solutions were prepared by 5–1000 fold dilution of the stock solution with water prior to HPLC analysis. Detection was accomplished with a diode array detector and chromatograms were recorded at 210 and 280 nm.1% orthophosphoric acid in methanol (v/v) (eluent B). and made to volume with the same solvent. Sencha tea was a present from Japan. Preparation of samples For tea leaves. dissolved in water by sonication for 1 min and made to volume with water and centrifuged as for tea leaves prior to HPLC analysis. For tea dry extract.4.1% orthophosphoric acid in water (v/v) (eluent A) and 0. and diode array detector was used. 2. (À)-epicatechin gallate [(À)-ECG]. This paper presents an alternative method of approaching the quantitative analysis of tea catechins by establishing response factors for single catechin standards using a gradient elution system. Hertfordshire. Methanol (HPLC grade) and orthophosphoric acid (analytical grade) were purchased from Fisher Scientific (Essex. and the flask and filter rinsed with the solution of ethanol/water (10:90. and the response factors for the catechins were calculated as a ratio of the concentration in mg/ml in relation to the corresponding area in the chromatogram. Relative response factors for individual catechins using different reference catechins are given. 2. Each sample was analysed in triplicate and the quantification of catechin content in the samples was carried out according to the following equation: Content ð%. and made to volume with water.5. Peaks were identified by comparing their retention times and UV spectra in the 200–400 nm range with authentic standards and by checking the purity of the peaks. (À)-gallocatechin gallate [(À)-GCG]. / Food Chemistry 81 (2003) 307–312 mobile phases and detectors (Merken & Beecher.6 mm) guard column. (À)-epicatechin [(À)-EC]. linear gradient from 40 to 50% B. about 0. The column used was a C18 reversed phase Kingsorb 5 mm (150Â4. dissolved in water by sonication for 1 min.3. 24% B. Preparation of catechin standard solutions Stock solution. 25–50 mg were accurately weighed into a 50 ml volumetric flask. Wang et al. linear gradient from 24 to 40% B. autosampler. Materials (+)-Gallocatechin [(+)-GC].25 g ground leaves were accurately weighed and extracted with 40 ml of a solution of ethanol/water (10:90. 7–10 min. Post-run time was 5 min. (À)-epigallocatechin gallate [(À)-EGCG]. Although most of the methods are practicable. 2.5 Mcm. . (À)-epigallocatechin [(À)-EGC].308 H. 2000). 10–20 min. approximately 10 mg of each catechin reference standard were accurately weighed into a 25-ml volumetric flask. w=wÞ ¼  à  à Asamp  RRF  Rf c  Vsamp  100 = Wsamp  1000 where: Asamp: area due to the catechins in the sample (mAU*s). Gunpowder tea and Ceylon black tea were purchased from a local teashop in Hitchin.

With 5 min of post-run for re-equilibration the column can be brought to the initial conditions ready for the next injection.8. and Fig. As can be Table 1 Response factors (RF) [(mg/ml)/mAU*s] for catechin standards and their relative response factors (RRF) at 210 nm Compound (+)-GC (À)-EGC (+)-C (À)-EGCG (À)-EC (À)-GCG (À)-ECG (À)-CG RF 0. Saijo & Takeda. Chromatogram of catechin standards. (À)-ECG. 3. 2.01031 0. Wang et al. Fig.82836 RRF/ (À)-EC 1. 11.9. theobromine.12778 1. For chromatographic conditions see Section 2. Response factors for all of the catechin standards together with their relative response factors against (+)-C.1..3. (À)-GCG.02606 RRF/ (+)-C 1.2. 2000).02870 0. 4. theobromine. similar results could be obtained using Genesis 5m C18 (150 Â 4.00984 0. 7. 9.99780 1.00948 0. caffeine.87063 1. Fig.. Fig. which were regarded as being present in samples below the detection limit when detected at 280 nm (Khokhar et al. (À)-EC.00986 0.14850 0.H. (+)-C.4 times larger than those at 280 nm for (+)-GC. 5. 1. 9. (À)-EC and (À)EGCG are readily available from various commercial sources. It can be seen from this that a good separation can be achieved within 25 min using the conditions described.96126 1. (À)-GCG. which takes too long to elute in an isocratic system. 1999).86873 0. 7.01015 0. (+)-GC.6. gallic acid.91058 0. 3. Chromatogram of a green tea extract. 8.00938 RRF/ (À)-EGCG 0. (À)-GCG. (À)-EC and (À)-EGCG at 210 and 280 nm are listed in Tables 1 and 2. 2 a typical sample of a green tea extract. (+)-C. Relative response factors of catechins Reference standards of (+)-C. Helliwell et al. (À)-EC.6 mm) columns. 10. a gradient elution system has been developed for the separation of catechins together with gallic acid.6.80736 0.03659 1. 69. (À)-CG. (À)-ECG. (À)-EGC.8. (À)-CG. (À)-EGCG. and 5. 7.07840 1.00000 1. 2000).07608 1.10926 1. 5. Helliwell et al. . (+)-C. (À)-EC.04581 0.83696 0. respectively. 1997.01133 0.23854 1. theobromine and caffeine. 15. Peak identification: 1. gallic acid. 8.00914 0. (À)-EGC. 2.89566 0. 16. 1 demonstrates the separation obtained for a mixture of reference standards. 3. Separation of catechins Based on our previous work on an isocratic elution system for the determination of catechins (Wang.. 5. (À)-EGCG. Peak identification: 1.92730 0. 6. 11.95147 seen the response factors at 210 nm were 56. 8. (À)-ECG and (À)-CG. This is especially useful for the determination of (+)-GC and (+)C. (+)-GC. (À)-EGCG.00000 0.6 mm) and Luna 5m C18 (2) (150 Â 4. / Food Chemistry 81 (2003) 307–312 309 3. Results 3. 10.00000 1. (À)-EGC. 6. For chromatographic conditions see Section 2. 4. 2. caffeine.2. It was found that using 210 nm as a detection wavelength the chromatograms could be considerably improved in signal-tonoise ratio (Wang. Although Figs. using the described elution system. respectively. and (À)CG. 1 and 2 were obtained using the Kingsorb column described.

310 H. when measured at 280 nm the sequence is quite different being (À)EGC > (+) -GC > (+) .15154 0.25 0.39814 0.163 0. Helliwell.9386 14. and after 2 days. respectively.48077 0. Garcia-Moreno. 6.878 used to investigate the possibility of using the method of relative response factors to analyse catechins. The characteristics of the calibration curves.2360 100.778Æ7.D. This demonstrated that after the standard solution was kept at ambient temperature for 1 day.57627 0.99999 0. Cuadros.391 6.51875 1. Table 3 shows the variation of relative response factors on these three columns.37175 0..48–68.53–74. Wang et al.50–72.40741 1.68577 1. are listed in Table 4.2071 110.C > (À)-EC > (À)-EGCG > (À) GCG > (À)-ECG> (À)-CG.86 99. LOL is determined by the following equation ´ ´ ´ (Garcia.59–82.3.63961 0. ´ Castro.48–68.31–44.86 99.258 0.008 Caffeine 5.99999 0.654 5.39367 1.2%.91 0.000 0. Rowe.74738 0.09243 0.841 6.65274 0.5225 À0.552 0.99999 0.00000 0.193 (À)-EC 0.99996 0. the square of correlation coefficient (R2) and on-line linearity (LOL) for each catechin.%) 99.438Æ15. For example after the standard solution was kept at either 5  C in a refrigerator or À10  C in a freezer for 10 days the response factors decreased by 1.11 R2 0. Natera. Selection of reference catechin Different reversed-phase C18 columns.33357 RRF/ (+)-C 3.23 0.433 5. Therefore caffeine is not considered to be a suitable reference compound.97257 0.06033 0. indicating that the selected reference catechins could be used on different columns.809 0. An isocratic elution system (Wang. in which the same detection wavelength of 280 nm was used. 1997.02817 0.1305 103. 2002): LOLð%Þ ¼ 100 À RSDðbÞ Under the described HPLC conditions.06908 0.5396 3.00000 1. including the range of linearity. 3.16 0.37175 Calculated based on the data from Saijo and Takeda (1999).1983 109.00000 0.602 (+)-C 0.795 0.274Æ0.538 0.59319 0. were Table 4 Characteristics of the calibration curves Compound (+)-GC (À)-EGC (+)-C (À)-EGCG (À)-EC (À)-GCG (À)-ECG (À)-CG Linear range (mg/ml) 0.1479 99. However.48077 1. & Barroso.791 0.99995 0.330Æ0.800Æ0.66 0.4.531Æ5.05055 RRF/ (À)-EGCG 6.796 0.15582 0.725Æ3. Table 3 Variations of RRF on three different columns (%) (À)-EGCG (+)-GC (À)-EGC (+)-C (À)-EGCG (À)-EC (À)-GCG (À)-ECG (À)-CG 0.04 99.393 1.888 1.77 99.45585 0.23441 7.80228 4.380 1. However.22–30.69822 4.160 1.701 6.388 1.99927 0.1295 87.00000 0.12500 1. Ales.44334 0. 2000) was also used to compare the relative response factors. by 12.405 5. having considered their stability and cost.370Æ0.539Æ3.9542 96.2235 À1.000 0. Validation of the method Calibration graphs for the catechins were constructed using seven levels of concentration which covered the concentration ranges expected in the various tea samples. namely Kingsorb.694Æ3. the response factor decreased by 6.9%. Caffeine was considered as a reference compound because of its low cost and high stability. as previously described.9180 8. the response factors for catechins are sequenced as (À)-EGCG > (À)ECG > (+)-GC > (+)-C5(À)-EGC > (À)-GCG5(À)CG5(À)-EC at 210 nm.12500 3.795 0.9532 À5.2431 InterceptÆS. (À)-EC and (+)-C was less than 2% for all columns.756Æ0.910Æ0. et al.38721 0. However.554 0. / Food Chemistry 81 (2003) 307–312 Table 2 Response factors (RF) [(mg/ml)/mAU*s] for catechin standards and their relative response factors (RRF) at 280 nm Compound (+)-GC (À)-EGC (+)-C (À)-EGCG (À)-EC (À)-GCG (À)-ECG (À)-CG a RF 0.79 99.000 1.181Æ0.77 SlopeÆS. the variations in relative response factors for catechins against caffeine were all greater than 5%.99997 0. (+)-C is the preferred catechin for determination of relative response factors.32441 RRF/ (+)-Ca 3.552 0. However.88 99. The variation in relative response factors against (À)-EGCG.970Æ0.00000 0.696Æ5.20 0.4%. The stability of the standard solution of (+)-C was investigated further. 3.75 99. 95. Roman.55 0. & Sierra.5 and 1. as shown in Table 3.6729 .674 0.68474 0.166 0. if the standard solution was kept at low temperature it was found to be stable.54690 RRF/ (À)-EC 3. Genesis and Luna.D.99996 Linearity (LOL. It was found that the responses and the relative response factors were very similar to that with the gradient elution system.7316 À0.73–102. The latter sequence is similar to that given by Sajio and Takeda (1999).077 5.359Æ2.60994 1.

17 1. This indicated a good base line separation.53 1.46 Sample 2.04 0.11 0. The recovery was determined by spiking a tea extract sample with three different addition of catechin standard solutions.20 0.92 0.74 0. Wang et al.08 1.89 Sencha 0.54 Keemun nd 0.24 3. Coefficients of variation for results obtained from different columns and even different HPLC instrument (for example.51 0.19 1.37 0.13 nd 0.09 nd 13.07 6.12 0.22 3.83 0.08 0.12 6. as is) Compound Black teas Ceylon (+)-GC (À)-EGC (+)-C (À)-EGCG (À)-EC (À)-GCG (À)-ECG (À)-CG Æ Catechins nd. It can be seen that the coefficients of variation for the standard solution for all analytes were within 1.61 0.74 19.16 0. As expected.10 nd 0.3 101. while for the sample solution.7 95. w/w. This is because.9 311 Table 6 Coefficients of variation analysis of standard and sample solutions (%) (n=7) Compound (+)-GC (À)-EGC (+)-C (À)-EGCG (À)-EC (À)-GCG (À)-ECG (À)-CG Standard 0.41 0.25 4.9 7.32 1.43 0.25 1.5 between gallic acid and (+)-GC. 3.03 12.07 LODapprox (mg/ml) 0.01 0. the limit of detection (LODapprox) is determined by the following equation: LODapprox ¼ 3ðSs =bÞà ½ðn À 2Þ=ðn À 1ފ1=2 Table 7 Content of catechins in tea leaves and green tea dry extracts (%.61 B 0.67 0.98 where RSD(b) is the relative standard deviation of the slope (expressed as a percentage).96 0.72 1.64 Recovery (%) 91.27 0.70 0. ´ According to an ALAMIN program (Garcia et al. Quantitative measurement of different tea samples Four green teas. 1997).53 0.5 99. and maximum Rs was 12. in black Green teas Longjing 0.13%..01 8.19 1.92 .55 0.18 0.73 0.22 0.57 where n is number of total measurements for each calibration set.46 0. green tea contained a much higher quantity of catechins than black tea. To test the precision of the assay method.13 0.26 1.02 11.90 3.10 0.04 nd 0. the coefficients of variation were within 2.19 LOQapprox (mg/ml) 1. The results are shown in Table 5. Table 6 summarises the results obtained.92 0.09 1. The limit of quantitation (LOQapprox) is calculated by replacing 3 with 10 in the above equation.58 0.9% have been obtained.6 between (+)-C and caffeine in a typical tea extract sample.5 1.68 Dry extracts A 2.01%.27 1. Varian ProStar HPLC) were less than 5%.17 1.18 7.55 0.23 38.46 2.13 0.32 nd 0. in which Ss is the residual standard deviation and b is the slope of the calibration curve.70 0.31 14.03 12.999) and on-line linearity (> 99%). It was found that the minimum Rs was > 2.16 0. analytical sensitivity (AS) is determined by the ratio of Ss/b.02 0. / Food Chemistry 81 (2003) 307–312 Table 5 Performance characteristics Compound (+)-GC (À)-EGC (+)-C (À)-EGCG (À)-EC (À)-GCG (À)-ECG (À)-CG AS (mg/ml) 0.71 0.16 0.08 RGT 0.15 28.71 0.68 0.03 0. two black teas and two green tea dry extracts were analysed using the determined relative response factors and the results are shown in Table 7. It can be seen that an excellent linearity was observed for all catechins in the range studied both in the terms of the correlation coefficients (R2 > 0.24 1.38 0. The selectivity of this method and the efficiency of the column are evaluated by the resolution (Rs) of two vicinal peaks.74 0.53 nd 1.95 0.69 1.10 0.81 0.42 1.5 and 106.H. It can be seen from the table that the limits are low enough to analyse all eight catechins in real tea samples. The results are presented in Table 5. Good recoveries between 88.22 0.3 0. 0.31 Gunpowder 0.87 0.5 3. not detected.93 0.33 2.0 91.5 106.5.0 93.8 88. Running a blank injection after the analysis of either standard or tea sample solutions showed no memory effect.70 3.03 0. the catechin standard solution and one of the samples to be analysed were injected seven times under the chromatographic conditions described above.12 6.

& Beecher.. 46... Food Chemistry. M.. (1999). Inhibition of carcinogenesis by tea: the evidence from experimental studies. Y.. J. M. R. A.. 152–160. Conclusions and discussion The reported assay method uses relative response factors and a gradient elution system for the determination of catechins in tea. Kiso. C.. X. & Stiller. (1994). 171–172. & Takeda. 749. Helliwell. 805. D. H. Tea flavonoids: their functions. (1997). Beijing. Zhang. The catechin content in green tea leaves was from 11. G. Castro. J. Simultaneous analysis of individual catechins and caffeine in green tea. Cuadros. H. most of the catechins have been altered to form theaflavins and theorubigins during black tea manufacture. References Alexis. P. F. & Jongen. & Nelson. Thomas. M.61% (w/w. A. Headspace solid-phase microextraction analysis of aroma compounds in vinegar. K. & Hsieh. A. & Bouwens. and in green tea dry extracts from 28. upon their reference standards used.. Wang et al. E. (2000)... which lie within expected ranges. Furthermore.57% (w/w. Hollman. 600–604. J.. Provan. (1998).. A. 115–121. Dalluge. C. H... 68. 92. ´ Natera. R. M. utilisation and analysis.. L. International Journal of Dermatology. H. Z. 761–770.. HPLC analysis of catechins in various kinds of green teas produced in Japan and abroad. P. Bronner. Setiawan. Y. Method for determining the content of catechins in tea infusions by high-performance liquid chromatography. Tea and health. The purity of most commercial catechin standards has been derived using HPLC normalisation methods and any potential water and/or solvent content tends to be ignored. L... Venema... Dreostic. (1998). J. B. L. & Beecher.. Q. Q.. F.. K. 37. Cheng. J. G. Y.. 37. Wang. J. and eight different catechins together with gallic acid. T. Measurement of food flavonoids by high-performance liquid chromatography: a review. M.68 to 13. D. Yu. (2000). Guo. L. S.. & Sierra. Determination of tea catechins. M. China: Press of Chinese Agricultural Acience. Z.. Yoshida. (1997). (1997). ALAMIN: a chemometric program to check analytical method performance and to assess the trueness by standard addition methodology. 411–424. 693–704. Journal of Chromatography A. Saijo. B. tea extracts and some tea containing products. M. Tea and health. Journal of Chromatography A. F. 16(7). L. RP-HPLC method for the determination of tea catechins. J. 691–692. Trends in Food Science & Technology. Wang. & You.. Dekker. Wargovich. Dalluge. (1997). tea. K. Lu. Journal of Agricultural and Food Chemistry. Z.. M.. M. Journal of Chromatography A. C. J. Cancer Letters. Li. R. The response factors for catechins reported in this paper were also based on their labelled purity. 114. Wang. (1997). (2000). M.92 to 38. & Chen. R. / Food Chemistry 81 (2003) 307–312 Balentine. Isocratic elution system for the determination of catechins. R. Wiseman. S. C. routine analysis. 261–267. Nelson.. V.. G. W. & Sander. It is ideal for rapid. 295–299. Yu. D.. C. Kurtz.. V. Nippon Shokuhin Kagaku Kogaku Kaishi. S. F. G. as is). G. B. 138–147. Rowe.. Y. 48(3). Critical Reviews in Food Science and Nutrition. & Helliwell. 38. Selection of column and gradient elution system for the separation of catechins in green tea using high-performance liquid chromatography. & Yang. 11. (1999). Merken. 881. J. R. C. Balentine. Roman. 967. Goto. could be determined simultaneously.312 H. There is now a requirement to establish absolute response factors for these catechins in order to ensure harmonisation when reporting catechin content.. M. 265–274. ´ ´ ´ Garcia. The chemistry of tea flavonoids. H. H. (2000). Garcia-Moreno. W. Journal of Chromatography A. E. V. Critical Reviews in Food Science and Nutrition. G. It should be noted that accepted practice is to assay tea. 577–599. C. (2001). W.. sensitive and accurate and is applicable to all kinds of tea. & Barroso. this method is simple.. & Nagashima.. Validation study. M. Jones. 735–743. Khokhar. International Journal of Cancer. Lu. R. S. C. The present paper has demonstrated a robust method for the quantification of catechins in tea and related products. C. 793.. caffeine and gallic acid in green tea using HPLC. Journal of Chromatography A. (2002). Protective effect of green tea on the risks of chronic gastritis and stomach cancer. L. Potential therapeutic applications of tea in dermatology. A. especially for those laboratories where catechin standards are not readily available. 37.. 137–142. I. Trends in Analytical Chemistry. C. .. Critical Reviews in Food Science and Nutrition.. With this method good repeatability of results was obtained. 4.. C. tea extracts and where applicable tea containing products by comparison with an accurately prepared reference solution of available catechins using the labelled purity of the catechins as the basis for quantification. (1996). theobromine and caffeine. as is). Ales. 381–385.