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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 dif-
ferent 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 ()-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
mobile phases and detectors (Merken & Beecher, 2000). 2.3. Preparation of catechin standard solutions
Although most of the methods are practicable, the high
cost and instability of some catechin reference standards Stock solution, approximately 10 mg of each catechin
have limited their application. reference standard were accurately weighed into a 25-ml
This paper presents an alternative method of volumetric flask, dissolved in water by sonication for 1
approaching the quantitative analysis of tea catechins min, and made to volume with water. Working standard
by establishing response factors for single catechin solutions were prepared by 5–1000 fold dilution of the
standards using a gradient elution system. The elution stock solution with water prior to HPLC analysis.
system employs simple mobile phases containing
methanol and water. Relative response factors for indi- 2.4. Analytical determinations
vidual catechins using different reference catechins are
given. By using these relative response factors catechin An HP 1100 series liquid chromatograph system
analysis can be carried out with only one selected cate- comprising vacuum degasser, quaternary pump, auto-
chin as a reference standard. sampler, thermostatted column compartment, and
diode array detector was used. The column used was a
C18 reversed phase Kingsorb 5 mm (1504.6 mm)
2. Materials and methods (phenomenex1, UK) with a Kingsorb 5 mm C18 (30
4.6 mm) guard column. Mobile phases consisted of
2.1. Materials 0.1% orthophosphoric acid in water (v/v) (eluent A)
and 0.1% orthophosphoric acid in methanol (v/v) (elu-
(+)-Gallocatechin [(+)-GC], (+)-catechin [(+)-C], ent B). The gradient was as follows: 0–5 min, 20% B; 5–
()-epicatechin [()-EC], ()-epigallocatechin 7 min, linear gradient from 20 to 24% B; 7–10 min, 24%
[()-EGC], ()-epigallocatechin gallate [()-EGCG], B; 10–20 min, linear gradient from 24 to 40% B; 20–25
()-gallocatechin gallate [()-GCG], ()-epicatechin min, linear gradient from 40 to 50% B. Post-run time
gallate [()-ECG], ()-catechin gallate [()-CG] and was 5 min. Elution was performed at a solvent flow rate
gallic acid, caffeine and theobromine were purchased of 1 ml/min. Detection was accomplished with a diode
from Sigma Chemical Co (Dorset, UK). Methanol array detector and chromatograms were recorded at 210
(HPLC grade) and orthophosphoric acid (analytical and 280 nm. The column was maintained at 30 C. The
grade) were purchased from Fisher Scientific (Essex, sample injection volume was 10 ml. Peaks were identified
UK). The water used in HPLC and sampling was pre- by comparing their retention times and UV spectra in
pared with a Super Purity Water System (Purite Ltd, the 200–400 nm range with authentic standards and by
England) with a resistivity over 17.5 Mcm. checking the purity of the peaks.
Roasted green tea (RGT) and Keemun black tea were
obtained from the Tea Research Institute, Chinese 2.5. Calculation of relative response factors and
Academy of Agricultural Sciences. Gunpowder tea and quantification of catechins
Ceylon black tea were purchased from a local teashop
in Hitchin, Hertfordshire, UK. Sencha tea was a present The standard solution was analysed under the condi-
from Japan. Tea dry extract was either produced from tions detailed above, and the response factors for the
green tea by the reporting company or purchased from catechins were calculated as a ratio of the concentration
other manufactures. in mg/ml in relation to the corresponding area in the
chromatogram. The relative response factors were cal-
2.2. Preparation of samples culated as the ratio of the response factor for each
catechin to that of the chosen reference catechin. Each
For tea leaves, about 0.25 g ground leaves were accu- sample was analysed in triplicate and the quantification
rately weighed and extracted with 40 ml of a solution of of catechin content in the samples was carried out
ethanol/water (10:90, v/v) by sonication for 20 min. The according to the following equation:
extraction solution was filtered into a 50 ml volumetric
flask, and the flask and filter rinsed with the solution of Content ð%; w=wÞ ¼
ethanol/water (10:90, v/v), and made to volume with the
same solvent. Approximately 1 ml of the sample solution Asamp RRF Rf c Vsamp 100 = Wsamp 1000
was centrifuged at 13,000 rpm for 10 min prior to
HPLC analysis. For tea dry extract, 25–50 mg were where: Asamp: area due to the catechins in the sample
accurately weighed into a 50 ml volumetric flask, (mAU*s); RRF: the average relative response factor of
dissolved in water by sonication for 1 min and made to that catechin to the reference catechin; Rfc: response factor
volume with water and centrifuged as for tea leaves of the catechin standard [(mg/ml)/mAU*s]; Vsamp: volume
prior to HPLC analysis. of sample solution (ml); Wsamp: sample weight (mg).
H. Wang et al. / Food Chemistry 81 (2003) 307–312 309
3. Results
Table 1
3.1. Separation of catechins Response factors (RF) [(mg/ml)/mAU*s] for catechin standards and
their relative response factors (RRF) at 210 nm
Based on our previous work on an isocratic elution
Compound RF RRF/ RRF/ RRF/
system for the determination of catechins (Wang, Helli- ()-EGCG ()-EC (+)-C
well et al., 2000), a gradient elution system has been
developed for the separation of catechins together with (+)-GC 0.01015 0.89566 1.10926 1.02870
()-EGC 0.00984 0.86873 1.07608 0.99780
gallic acid, theobromine and caffeine. Fig. 1 demon-
(+)-C 0.00986 0.87063 1.07840 1.00000
strates the separation obtained for a mixture of refer- ()-EGCG 0.01133 1.00000 1.23854 1.14850
ence standards, and Fig. 2 a typical sample of a green ()-EC 0.00914 0.80736 1.00000 0.92730
tea extract. It can be seen from this that a good separa- ()-GCG 0.00948 0.83696 1.03659 0.96126
tion can be achieved within 25 min using the conditions ()-ECG 0.01031 0.91058 1.12778 1.04581
()-CG 0.00938 0.82836 1.02606 0.95147
described. With 5 min of post-run for re-equilibration
the column can be brought to the initial conditions
ready for the next injection. Although Figs. 1 and 2
were obtained using the Kingsorb column described, seen the response factors at 210 nm were 56.8, 69.6,
similar results could be obtained using Genesis 5m C18 15.8, 8.2, 16.6, 7.3, 5.9, and 5.4 times larger than those
(150 4.6 mm) and Luna 5m C18 (2) (150 4.6 mm) at 280 nm for (+)-GC, ()-EGC, (+)-C, ()-EGCG,
columns. ()-EC, ()-GCG, ()-ECG and ()-CG, respectively,
using the described elution system. It was found that
3.2. Relative response factors of catechins using 210 nm as a detection wavelength the chromato-
grams could be considerably improved in signal-to-
Reference standards of (+)-C, ()-EC and ()- noise ratio (Wang, Helliwell et al., 2000). This is espe-
EGCG are readily available from various commercial cially useful for the determination of (+)-GC and (+)-
sources. Response factors for all of the catechin stan- C, which were regarded as being present in samples
dards together with their relative response factors below the detection limit when detected at 280 nm
against (+)-C, ()-EC and ()-EGCG at 210 and 280 (Khokhar et al., 1997; Saijo & Takeda, 1999), and ()-
nm are listed in Tables 1 and 2, respectively. As can be CG, which takes too long to elute in an isocratic system.
Fig. 1. Chromatogram of catechin standards. For chromatographic conditions see Section 2. Peak identification: 1. gallic acid, 2. (+)-GC, 3. theo-
bromine, 4. ()-EGC, 5. (+)-C, 6. caffeine, 7. ()-EGCG, 8. ()-EC, 9. ()-GCG, 10. ()-ECG, 11. ()-CG.
Fig. 2. Chromatogram of a green tea extract. For chromatographic conditions see Section 2. Peak identification: 1. gallic acid, 2. (+)-GC, 3. theo-
bromine, 4. ()-EGC, 5. (+)-C, 6. caffeine, 7. ()-EGCG, 8. ()-EC, 9. ()-GCG, 10. ()-ECG, 11. ()-CG.
310 H. Wang et al. / Food Chemistry 81 (2003) 307–312
Table 4
Characteristics of the calibration curves
Table 7
Content of catechins in tea leaves and green tea dry extracts (%, w/w, as is)
tea, most of the catechins have been altered to form Balentine, D. (1997). Tea and health. Critical Reviews in Food Science
theaflavins and theorubigins during black tea manu- and Nutrition, 37, 691–692.
Balentine, D. A., Wiseman, S. A., & Bouwens, L. C. M. (1997). The
facture. The catechin content in green tea leaves was
chemistry of tea flavonoids. Critical Reviews in Food Science and
from 11.68 to 13.57% (w/w, as is), and in green tea dry Nutrition, 37, 693–704.
extracts from 28.92 to 38.61% (w/w, as is), which lie Bronner, W. E., & Beecher, G. R. (1998). Method for determining the
within expected ranges. content of catechins in tea infusions by high-performance liquid
chromatography. Journal of Chromatography A, 805, 137–142.
Cheng, Q. K., & Chen, Z. M. (1994). Tea and health. Beijing, China:
Press of Chinese Agricultural Acience.
4. Conclusions and discussion Dalluge, J. J., & Nelson, B. C. (2000). Determination of tea catechins.
Journal of Chromatography A, 881, 411–424.
The reported assay method uses relative response Dalluge, J. J., Nelson, B. C., Thomas, J. B., & Sander, L. C. (1998).
factors and a gradient elution system for the determi- Selection of column and gradient elution system for the separation
nation of catechins in tea. It is ideal for rapid, routine of catechins in green tea using high-performance liquid chromato-
graphy. Journal of Chromatography A, 793, 265–274.
analysis, especially for those laboratories where catechin Dreostic, I. E., Wargovich, M. J., & Yang, C. S. (1997). Inhibition of
standards are not readily available. With this method carcinogenesis by tea: the evidence from experimental studies. Cri-
good repeatability of results was obtained, and eight dif- tical Reviews in Food Science and Nutrition, 37, 761–770.
ferent catechins together with gallic acid, theobromine Garćia, A. M., Cuadros, L., Alés, F., Román, M., & Sierra, J. L.
and caffeine, upon their reference standards used, could be (1997). ALAMIN: a chemometric program to check analytical
method performance and to assess the trueness by standard addition
determined simultaneously. Furthermore, this method is methodology. Trends in Analytical Chemistry, 16(7), 381–385.
simple, sensitive and accurate and is applicable to all kinds Goto, T., Yoshida, Y., Kiso, M., & Nagashima, H. (1996). Simul-
of tea, tea extracts and some tea containing products. taneous analysis of individual catechins and caffeine in green tea.
It should be noted that accepted practice is to assay Journal of Chromatography A, 749, 295–299.
tea, tea extracts and where applicable tea containing Khokhar, S., Venema, D., Hollman, P. C. H., Dekker, M., & Jongen,
W. (1997). RP-HPLC method for the determination of tea cate-
products by comparison with an accurately prepared chins. Cancer Letters, 114, 171–172.
reference solution of available catechins using the label- Merken, H. M., & Beecher, G. R. (2000). Measurement of food
led purity of the catechins as the basis for quantifi- flavonoids by high-performance liquid chromatography: a review.
cation. The response factors for catechins reported in Journal of Agricultural and Food Chemistry, 48(3), 577–599.
Natera, R., Castro, R., Garćia-Moreno, M. V., Rowe, F. G., & Bar-
this paper were also based on their labelled purity. The
roso, C. G. (2002). Headspace solid-phase microextraction analysis
purity of most commercial catechin standards has been of aroma compounds in vinegar. Validation study. Journal of
derived using HPLC normalisation methods and any Chromatography A, 967, 261–267.
potential water and/or solvent content tends to be Saijo, R., & Takeda, Y. (1999). HPLC analysis of catechins in various
ignored. The present paper has demonstrated a robust kinds of green teas produced in Japan and abroad. Nippon Shokuhin
method for the quantification of catechins in tea and Kagaku Kogaku Kaishi, 46, 138–147.
Setiawan, V. W., Zhang, Z. F., Yu, G. P., Lu, Q. Y., Li, Y. L., Lu,
related products. There is now a requirement to establish M. L., Wang, M. R., Guo, C. H., Yu, S. Z., Kurtz, R. C., & Hsieh,
absolute response factors for these catechins in order to C. C. (2001). Protective effect of green tea on the risks of chronic
ensure harmonisation when reporting catechin content. gastritis and stomach cancer. International Journal of Cancer, 92,
600–604.
Wang, H., Helliwell, K., & You, X. (2000). Isocratic elution system for
References the determination of catechins, caffeine and gallic acid in green tea
using HPLC. Food Chemistry, 68, 115–121.
Alexis, A. F., Jones, V. A., & Stiller, M. J. (1999). Potential ther- Wang, H., Provan, G. J., & Helliwell, K. (2000). Tea flavonoids: their
apeutic applications of tea in dermatology. International Journal of functions, utilisation and analysis. Trends in Food Science & Tech-
Dermatology, 38, 735–743. nology, 11, 152–160.