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Food Chemistry 81 (2003) 307–312

www.elsevier.com/locate/foodchem

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 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

1. Introduction bioactive compounds in tea, are responsible for the


claimed therapeutic activity of tea.
Tea is the most popular beverage after water In addition to the direct consumption of tea either by
throughout the world. Tea was first used in China for its brewing loose leaves or tea bags or in a ready-to-drink
medicinal properties 5000 years ago (Balentine, 1997; form, in recent years, there have been more and more
Cheng & Chen, 1994), but it is only in the last couple of applications for tea extracts especially in the nutra-
decades that the potential health benefits of this ancient ceutical, and food areas. Therefore it is essential to be
beverage have been documented on a scientific basis. A able to offer consumers a consistent level of catechins in
growing body of evidence from both human and animal their products. Tea leaves, their extracts and the con-
studies suggests that regular consumption of green tea sumer products themselves need to be standardised and
can reduce the incidence of a variety of cancers, including routinely assayed. Furthermore, any potential health
colon, pancreatic, and stomach cancers, and other dis- implications require data generation on the content of
eases (Alexis, Jones, & Stiller, 1999; Balentine, Wise- catechins in tea leaves, their extracts and products. For
man, & Bouwens, 1997; Dreostic, Wargovich, & Yang, these purposes, much attention has been paid to devel-
1997; Steiawan et al., 2001; Wang, Provan, & Helliwell, oping analytical methods for tea catechins (Bronner &
2000). It is generally believed that catechins, the principle Beecher, 1998; Dalluge & Nelson, 2000; Dalluge, Nel-
son, Thomas, & Sander, 1998; Goto, Yoshida, Kiso, &
Nagashima, 1996; Khokhar, Venema, Hollman, Dek-
* Corresponding author. Tel.: +44-1462-437615; fax: +44-1462-
ker, & Jongen, 1997; Wang, Helliwell, & You, 2000).
420528. Analysis of catechins is generally accomplished by
E-mail address: hwang@williamransom.com (H. Wang). 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
308 H. Wang et al. / Food Chemistry 81 (2003) 307–312

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 2 used to investigate the possibility of using the method of


Response factors (RF) [(mg/ml)/mAU*s] for catechin standards and relative response factors to analyse catechins. Table 3
their relative response factors (RRF) at 280 nm
shows the variation of relative response factors on these
Compound RF RRF/ RRF/ RRF/ RRF/ three columns. The variation in relative response factors
()-EGCG ()-EC (+)-C (+)-Ca against ()-EGCG, ()-EC and (+)-C was less than
(+)-GC 0.57627 6.23441 3.80228 3.69822 3.12500
2% for all columns, indicating that the selected refer-
()-EGC 0.68474 7.40741 4.51875 4.39367 3.12500 ence catechins could be used on different columns.
(+)-C 0.15582 1.68577 1.02817 1.00000 1.00000 However, having considered their stability and cost,
()-EGCG 0.09243 1.00000 0.60994 0.59319 0.48077 (+)-C is the preferred catechin for determination of
()-EC 0.15154 1.63961 1.00000 0.97257 1.00000 relative response factors. Caffeine was considered as a
()-GCG 0.06908 0.74738 0.45585 0.44334 0.48077
()-ECG 0.06033 0.65274 0.39814 0.38721 0.37175
reference compound because of its low cost and high
()-CG 0.05055 0.54690 0.33357 0.32441 0.37175 stability. However, as shown in Table 3, the variations
in relative response factors for catechins against caffeine
a
Calculated based on the data from Saijo and Takeda (1999). were all greater than 5%. Therefore caffeine is not con-
sidered to be a suitable reference compound.
Table 3 The stability of the standard solution of (+)-C was
Variations of RRF on three different columns (%) investigated further. This demonstrated that after the
standard solution was kept at ambient temperature for 1
()-EGCG ()-EC (+)-C Caffeine
day, the response factor decreased by 6.9%, and after 2
(+)-GC 0.674 0.809 0.796 5.654 days, by 12.2%. However, if the standard solution was
()-EGC 0.388 0.552 0.795 5.841 kept at low temperature it was found to be stable. For
(+)-C 1.166 0.552 0.000 6.433 example after the standard solution was kept at either 5  C
()-EGCG 0.000 0.791 1.163 5.701
()-EC 0.795 0.000 0.554 6.391
in a refrigerator or 10  C in a freezer for 10 days the
()-GCG 0.888 0.160 0.393 6.405 response factors decreased by 1.5 and 1.4%, respectively.
()-ECG 1.258 1.538 1.380 5.077 An isocratic elution system (Wang, Helliwell, et al.,
()-CG 0.193 0.602 1.008 5.878 2000) was also used to compare the relative response
factors. It was found that the responses and the relative
response factors were very similar to that with the gra-
dient elution system.
Under the described HPLC conditions, the response
factors for catechins are sequenced as ()-EGCG > ()- 3.4. Validation of the method
ECG > (+)-GC > (+)-C5()-EGC > ()-GCG5()-
CG5()-EC at 210 nm. However, when measured at Calibration graphs for the catechins were constructed
280 nm the sequence is quite different being ()- using seven levels of concentration which covered the
EGC > (+) -GC > (+) - C > ()-EC > ()-EGCG > () - concentration ranges expected in the various tea samples.
GCG > ()-ECG> ()-CG. The latter sequence is simi- The characteristics of the calibration curves, including the
lar to that given by Sajio and Takeda (1999), in which range of linearity, the square of correlation coefficient (R2)
the same detection wavelength of 280 nm was used. and on-line linearity (LOL) for each catechin, are listed in
Table 4. LOL is determined by the following equation
3.3. Selection of reference catechin (Garćia, Cuadros, Alés, Román, & Sierra, 1997; Natera,
Castro, Garćia-Moreno, Rowe, & Barroso, 2002):
Different reversed-phase C18 columns, namely King-
sorb, Genesis and Luna, as previously described, were LOLð%Þ ¼ 100  RSDðbÞ

Table 4
Characteristics of the calibration curves

Compound Linear range R2 Linearity SlopeS.D. InterceptS.D.


(mg/ml) (LOL,%)

(+)-GC 0.48–68.66 0.99995 99.75 95.9700.2360 6.5315.9180


()-EGC 0.73–102.23 0.99997 99.79 100.3700.2071 8.7787.7316
(+)-C 0.48–68.16 0.99999 99.88 110.8000.1295 0.7253.2235
()-EGCG 0.59–82.20 0.99996 99.77 87.1810.1983 1.6965.9532
()-EC 0.50–72.91 0.99999 99.86 109.3300.1479 5.6943.9386
()-GCG 0.31–44.55 0.99927 99.04 99.7560.9542 14.43815.5225
()-ECG 0.53–74.25 0.99999 99.86 96.2740.1305 0.5393.5396
()-CG 0.22–30.11 0.99996 99.77 103.9100.2431 3.3592.6729
H. Wang et al. / Food Chemistry 81 (2003) 307–312 311

Table 5 where n is number of total measurements for each cali-


Performance characteristics bration set. The limit of quantitation (LOQapprox) is
Compound AS LODapprox LOQapprox Recovery calculated by replacing 3 with 10 in the above equation.
(mg/ml) (mg/ml) (mg/ml) (%) The results are shown in Table 5. It can be seen from the
table that the limits are low enough to analyse all eight
(+)-GC 0.16 0.46 1.53 91.8
catechins in real tea samples.
()-EGC 0.20 0.58 1.92 88.5
(+)-C 0.08 0.22 0.72 99.0 To test the precision of the assay method, the catechin
()-EGCG 0.18 0.51 1.70 91.3 standard solution and one of the samples to be analysed
()-EC 0.10 0.27 0.90 101.0 were injected seven times under the chromatographic
()-GCG 0.41 1.16 3.87 93.7 conditions described above. Table 6 summarises the
()-ECG 0.10 0.27 0.92 95.5
results obtained. It can be seen that the coefficients of
()-CG 0.07 0.19 0.64 106.9
variation for the standard solution for all analytes were
within 1.01%, while for the sample solution, the coeffi-
cients of variation were within 2.13%. Coefficients of
Table 6 variation for results obtained from different columns
Coefficients of variation analysis of standard and sample solutions (%) and even different HPLC instrument (for example,
(n=7) Varian ProStar HPLC) were less than 5%.
Compound Standard Sample The selectivity of this method and the efficiency of the
column are evaluated by the resolution (Rs) of two vic-
(+)-GC 0.73 2.13
()-EGC 0.83 0.19
inal peaks. It was found that the minimum Rs was
(+)-C 0.32 1.25 > 2.5 between gallic acid and (+)-GC, and maximum
()-EGCG 1.01 1.33 Rs was 12.6 between (+)-C and caffeine in a typical
()-EC 0.81 2.08 tea extract sample. This indicated a good base line
()-GCG 0.61 1.26 separation.
()-ECG 0.70 1.42
()-CG 0.46 1.98
The recovery was determined by spiking a tea extract
sample with three different addition of catechin stan-
dard solutions. The results are presented in Table 5.
Good recoveries between 88.5 and 106.9% have been
where RSD(b) is the relative standard deviation of the obtained.
slope (expressed as a percentage). It can be seen that an Running a blank injection after the analysis of either
excellent linearity was observed for all catechins in the standard or tea sample solutions showed no memory
range studied both in the terms of the correlation coef- effect.
ficients (R2 > 0.999) and on-line linearity (> 99%).
According to an ALAMIN program (Garćia et al., 3.5. Quantitative measurement of different tea samples
1997), analytical sensitivity (AS) is determined by the ratio
of Ss/b, in which Ss is the residual standard deviation and Four green teas, two black teas and two green tea dry
b is the slope of the calibration curve; the limit of detection extracts were analysed using the determined relative
(LODapprox) is determined by the following equation: response factors and the results are shown in Table 7.
As expected, green tea contained a much higher quan-
LODapprox ¼ 3ðSs =bÞ ½ðn  2Þ=ðn  1Þ
1=2 tity of catechins than black tea. This is because, in black

Table 7
Content of catechins in tea leaves and green tea dry extracts (%, w/w, as is)

Compound Black teas Green teas Dry extracts

Ceylon Keemun Sencha Longjing RGT Gunpowder A B

(+)-GC 0.03 nd 0.25 0.17 0.24 0.22 2.01 0.9


()-EGC 0.12 0.10 4.96 1.22 3.37 3.03 8.04 7.55
(+)-C 0.02 nd 0.12 0.18 0.12 0.07 0.74 0.31
()-EGCG 0.71 0.43 6.13 7.74 6.53 6.11 19.46 14.5
()-EC 0.13 0.04 0.93 0.55 0.67 0.68 2.69 1.71
()-GCG nd nd 0.09 0.24 0.19 0.17 1.70 0.5
()-ECG 0.53 0.32 1.09 1.95 1.16 1.38 3.74 3.3
()-CG nd nd nd 0.03 0.03 0.02 0.23 0.15
 Catechins 1.54 0.89 13.57 12.08 12.31 11.68 38.61 28.92

nd, not detected.


312 H. Wang et al. / Food Chemistry 81 (2003) 307–312

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