Food Chemistry 172 (2015) 469475

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Simultaneous determination of 15 phenolic compounds and caffeine

in teas and mate using RP-HPLC/UV detection: Method development
and optimization of extraction process
In Kyung Bae a, Hyeon Mi Ham b, Min Hee Jeong b, Dong Ho Kim b, Ho Jin Kim b,
a
b

School of Applied Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea
National Agricultural Products Quality Management Service, Seoul 150-804, Republic of Korea

a r t i c l e

i n f o

Article history:
Received 9 September 2013
Received in revised form 25 August 2014
Accepted 10 September 2014
Available online 17 September 2014
Keywords:
RP-HPLC/UV
Response surface methodology
Validation
Phenolic compounds
Caffeine
Tea

a b s t r a c t
A reversed-phase high performance liquid chromatographic coupled to ultraviolet detection (RP-HPLC/
UV) method was developed for simultaneous determination of 15 phenolic compounds and caffeine in
TEAS (green tea, oolong tea, black tea and mate). Furthermore, the extraction process of total phenolic
contents (TPC) from TEAS were optimized using response surface methodology (RSM) based on a central
composite design (CCD) and then applied to extraction of TEAS. The best conditions obtained using the
model were as follow: green tea extraction time of 123 min, extraction temperature of 70 C and
ethanol concentration of 75%, oolong tea extraction time of 98 min, extraction temperature of 70 C
and ethanol concentration of 69%, black tea extraction time of 105 min, extraction temperature of
71 C and ethanol concentration of 63%, and mate extraction time of 103 min, extraction temperature
of 71 C and ethanol concentration of 61%. Among the extraction methods used in this study, heat-reux
extraction was found to result in the highest values of TPC. The chromatographic peaks of the 16 studied
compounds were successfully identied by comparing their retention time and UV spectra with the
reference standards. Method validation was performed by means of linearity, sensitivity, selectivity,
accuracy and precision. The developed method was found to be simple, specic and reliable and is suited
for routine analysis of phenolic compounds and caffeine in TEAS.
2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction
Tea is undoubtedly one of the most popular and widely consumed beverages in the world. It gained its popularity mostly by
attractive aromas and avours, and appears to become more popular as its health benets revealed. In numerous epidemiological
studies, it is also revealed that tea consumption may reduce the
risk of a variety of the risk of cancers and cardiovascular diseases
(Horzic et al., 2009; Kuzuhara, Suganuma, & Fujiki, 2008). Moreover, the biological functions of tea have also been reported, such
as anti-inammation, anti-oxidation, anti-allergy, and anti-obesity
(Hu et al., 2009). These health benets of tea mainly come from its
high content of phenolic compounds, particularly the catechin
derivatives. It also contains quite an amount of caffeine. On average, tea leaves contain 3% caffeine by weight, although this can
range from 2.0% to 6.9% (Rahim, Nofrizal, & Saad, 2014). It is tend
to increase alertness, reaction times and mitigate tiredness by
stimulating the central nervous system (Grifths & Grifths,
Corresponding author. Tel.: +82 2 2165 6141; fax: +82 2 2165 6008.
E-mail address: rex7878@korea.kr (H.J. Kim).

2005). Therefore, it is required to develop a proper method for

simultaneous determination of the phenolic compounds and caffeine in tea to investigate their qualities and health promoting
properties.
To date, various methods have been reported for the characterization of phenolic compounds and caffeine in tea, such as high
performance liquid chromatography (HPLC) coupled with ultraviolet detection (UV) (Hadad, Salam, Soliman, & Mesbah, 2012), electrochemical detection (ECD) (Novak, Seruga, & Komorsky-Lovric,
2010), mass spectrometry (MS) (Fraser et al., 2012; Wu, Xu,
Hritier, & Andlauer, 2012) or particle beam/electron ionization
mass spectrometry (PB/EIMS) (Castro, Pregibon, Chumanov, &
Marcus, 2010); gas chromatography coupled with MS (Shadkami,
Estevez, & Helleur, 2009); high-speed counter-current chromatography (HSCCC) (Kumar, Wijekoon, Kumar, Punyasiri, & Abeysinghe,
2009); chiral capillary electrophoresis (CCE) (El-Hady & El-Maali,
2008); Fourier transform near infrared reectance (FT-NIR) spectroscopy (Chen, Zhao, Chaitep, & Guo, 2009). The most commonly
used method for the determination of tea components is based
on the HPLC separation (Qi, Zeng, Wen, Liang, & Zhang, 2011).
HPLCMS and HPLCMS/MS, which provide information about

http://dx.doi.org/10.1016/j.foodchem.2014.09.050
0308-8146/ 2014 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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I.K. Bae et al. / Food Chemistry 172 (2015) 469475

the molecular mass and structural features of components, are

considered to be more useful than other methods in separation,
identication and quantication of the characteristic compounds
in tea. However, since these methods are quite costly to purchase
and maintain, many laboratories prefer to use HPLCUV detection,
which found to be less costly, comparably convenient to operate,
and suitable for routine analysis, for the determination of phenolic
compounds and caffeine in TEAS (green tea, oolong tea and black
tea; Camellia sinensis and mate; Ilex paraguariensis) (Magiera
et al., 2011; Rahim et al., 2014).
Extraction is the initial process, and it is also an essential step in
the recovery and purication of phenolic compounds from plant
materials (Prasad et al., 2011). The efciency of extraction depends
on several parameters, such as temperature, time, solvent polarity,
and pH, and their effects can be either independent or interactive
(Murakami et al., 2011). Response surface methodology (RSM) is
a useful statistical technique for evaluating the effects of multiple
factors and their interactions, and it is also can be effectively used
for nding combinations of these factors, which will produce the
optimum response (Chiste, Yamashita, Gozzo, & Mercadante,
2011). The main advantage of this technology is the reduced number of experimental trials that are needed to evaluate multiple factors and their interactions, thus it is found to be less laborious and
time-consuming than other optimization approaches (e.g., the onevariable-at-a-time optimization) (Frys, Cesla, Adam, & Ventura,
2012; Jeong, Kwak, Ahn, & Jeong, 2012). Central composite design
(CCD) is the most popular form of RSM and it has been widely used
for analysis to optimize various parameters in food processing,
such as extraction and manufacturing conditions (Chiste et al.,
2011; Lasekan & Abbas, 2011).
In this study, we described the development and validation of a
simple and reliable method for simultaneous separation, identication and quantication of 15 phenolic compounds and caffeine
in TEAS using reversed-phase high performance liquid chromatography coupled to ultraviolet detection (RP-HPLC/UV). Extraction
conditions were optimized by RSM based on a CCD to obtain the
maximum extraction yields of total phenolics from TEAS.
2. Materials and methods
2.1. Chemicals and reagents
HPLC-grade acetonitrile, methanol, and ethanol were purchased
from Merck (Darmstadt, Germany), and acetic acid was purchased
from Kanto (Tokyo, Japan). The Folin & Ciocalteus phenol reagent
and sodium carbonate were obtained from SigmaAldrich (St.
Louis, MO, USA). Water was puried using a Milli-Q Rios/Elix water
purication system (Millipore, Bedford, MA, USA). Standards of
Catechins (epicatechin, gallocatechin, epigallocatechin, epigallocatechin gallate, epicatechin gallate, gallocatechin gallate and catechin gallate), rutin, quercetrin, gallic acid, 3-hydroxy benzoic
acid, caffeic acid, chlorogenic acid, ferulic acid, sinapic acid and caffeine were purchased from SigmaAldrich (St. Louis, MO, USA).
2.2. Chemicals and reagents
Samples of TEAS were purchased from a local supermarket. The
four dried samples were then pulverized into ne powders with a
pulverizer (HMF-100; HANIL Electric Co., Seoul, Korea). The pulverizer was used by setting it to a maximum speed of 22,000 rpm. In
addition, the ne powders were generally ranged from 400 lm to
1000 lm in size.
To nd out an optimum extraction method for TEAS samplings,
the efciency of four different extraction methods (heat-reux
extraction, ultrasonic-assisted extraction, microwave-assisted

extraction and shaking extraction) was investigated by applying

them to the samplings. The same extraction time (90 min), ethanol
concentration (50%) and solid-solvent ratio (1:50) were applied in
all extractions.
Heat-reux extraction (HRE) was performed using a reux condenser (Digital extraction, ROTA-MS-EAMD, Daejeon, Korea) with a
magnetic stirrer. 2 g of each powdered sample from TEAS was
accurately weighted and added into a 100 mL round bottom ask,
and then extracted with 100 mL. Ultrasonic-assisted extraction
(UAE) was carried out using an ultrasonic cleaning bath (S450H;
HUCOM system, Suwon, Korea) by adding 2 g of each sample into
a 100 mL volumetric ask and operating at 70 C. Microwaveassisted extraction (MAE) was conducted in a microwave digestion
system (Start D; Milestone Srl, Italy) at a nominal power of 1000 W
and extraction temperature at 70 C by mixing the same amount of
powder and ethanol into a Teon extraction vessel (100 mL). Shaking extraction (SE) was also performed by placing a conical ask,
which contained 0.5 g of tea powder and 25 mL of 50% ethanol
on a shaker (SR-2W; Taitec, Saitama, Japan), and operating at a
high speed (Set # 10) at room temperature. All extractions were
repeated three times and ltered through 0.45 lm disposable lters before chemical analysis.
2.3. Determination of total phenolic contents
The total phenolic contents (TPC) of the TEAS extracts were
determined according to the literatures (Fu et al., 2011; Singleton
& Rossi, 1965). Briey, 0.5 mL of the appropriate dilution of the
four extracts (Section 2.2) were mixed 2.5 mL of 1:10 diluted
FolinCiocalteu reagent and allowed to stand for 4 min. Subsequently, 2 mL of saturated sodium carbonate solution was mixed
together and allowed to stand for 2 h. And then the sample absorbance was measured by spectrophotometer (Multiskan GO,
Thermo Scientic, Vantaa, Finland) at 760 nm. Since gallic acid is
one of the most abundant polyphenols found in tea (Dulce,
Pedro, Maria, & Maria, 2011; Elisabetta, Tiziana, Lucia, Luca, &
Patricia, 2014), several studies have used it to calculate TPC values
(Kim, Goodner, Park, Choi, & Talcott, 2011; Oh, Jo, Cho, Kim, & Han,
2013; Ramalho et al., 2013). For this reason, TPC was expressed as
milligrams of gallic acid equivalent (GAE) per gram of TEAS. All
measurements were performed in triplicate.
2.4. Preparation of working standard solutions
15 phenolic compounds and caffeine were dissolved with methanol in a 10 mL volumetric ask to produce stock standard solutions. The concentrations of stock solutions ranged from were 1.0
to 10 mg/mL. The appropriate volumes of each stock solution were
mixed together, and then diluted serially to prepare the working
standard solutions. All solutions were stored under refrigeration.
2.5. HPLC analysis
HPLC analysis was performed on a Waters ACQUITY UPLC
H-Class system (Waters, Milford, MA, USA) equipped with a binary
solvent delivery pump, an auto sampler, and a photodiode array
detector (PDA) and controlled by the Empower-II software.
A reversed phase Scherzo SS-C18 (150 mm  4.6 mm i.d., 3 lm
particle size) column was used for all separations at a column temperature of 30 C. The mobile phase composed of A (1% acetic acid
in acetonitrile) and B (1% acetic acid in water) with gradient elution: 0 min (5% A), 021 min (520% A), 2130 min (2025% A),
3032 min (25100% A), 3239 min (100100% A), 3940 min
(1005% A), and 4045 min (55% A) was used in this study. The
sample injection volume was 5 lL, and the ow-rate was set at
0.4 mL/min. Peaks were detected at 270 nm (epicatechin,

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Table 1
Central composite design: independent variable levels (origin and coded).
Independent variable

Table 2
Comparison of the TPC obtained by extraction with different methods, including HRE,
UAE, MAE and SE.

Level
2

Time (min)
Temperature (C)
Ethanol concentration (%)

X1
X2
X3

1

30
50
10

60
60
30

0
90
70
50

1
120
80
70

Matrix

2
150
90
90

gallocatechin, epigallocatechin, epigallocatechin gallate, epicatechin gallate, gallocatechin gallate, catechin gallate, rutin, quercetrin, gallic acid, 3-hydroxy benzoic acid and caffeine) and 320 nm
(caffeic acid, chlorogenic acid, ferulic acid and sinapic acid) and
applied for validation of method and analysis. The mobile phase
was ltered through a 0.45 lm membrane lter (Millipore, Milford, MA, USA) and degassed under vacuum.
2.6. Method validation
Method validation was performed according to guidelines set
by International Conference on Harmonisation (ICH (Q2), 2005)
and International Union of Pure and Applied Chemistry (IUPAC,
2002). The method was validated in terms of linearity, sensitivity,
selectivity, accuracy and precision.
2.7. Experimental design
The optimized conditions for the extraction of TPC from TEAS
were applied by RSM and analysed using MINITAB Release 14.
(Minitab Korea, Gunpo, RK). Based on the screening results, a
CCD with 5 levels and 3 factors (extraction time, extraction temperature, and ethanol concentration) was used. Detailed data are
presented in Table 1. 20 experimental settings were consisted of
a 23 factorial design with 6 axial points and 6 repetitions at the
central point. A mathematical regression model for the response
of Y (the predicted response) was tted in Eq. (1) as follow:

Y b0

Xa

b Xi
i1 i

Xa

b X2
i1 ii i

Xa

b XiXj
i;j1ij ij

where Y is response (TPC, (%), b0 is the coefcient constant, bi is the

linear coefcient, bii is the coefcient of squared effect, bji is the
coefcient of interaction effect, and Xi and Xj are the coded values
of variables i and j, respectively (extraction time (X1), extraction
temperature (X2), and ethanol concentration (X3)).
3. Results and discussion
3.1. Experimental design and optimization of extraction conditions
using RSM
There are a number of factors to be considered to nd optimum
experimental conditions of sample preparation. In this study, RSM
was employed with a CCD to determine the optimum conditions
for extraction. We also tried to identify the interactions between
parameters, as well as the linear relationships between response
variables. Before optimizing extraction parameters with CCD, a
preliminary experiment has been performed.
To investigate the optimal extraction method for TEAS, a comparison of the TPC obtained by four common extraction methods
(HRE, UAE, MAE and SE) was performed. In the extraction, different
temperature settings were applied as 70 C for HRE, UAE and MAE,
and room temperature for SE. But extraction time (90 min), ethanol
concentration (50%) and solidsolvent ratio (1:50) were remained
identical for all extractions. Table 2 shows that the TPC was found
to be higher when TEAS were extracted by HRE compared to that of

Green tea
Oolong tea
Black tea
Mate
a

TPC
HRE
Mean SDa

UAE
Mean SD

MAE
Mean SD

SE
Mean SD

67.5 1.1
50.6 1.2
27.0 0.8
31.1 1.2

51.3 1.7
45.1 1.2
21.0 2.0
21.5 1.4

25.0 1.5
22.5 1.5
13.5 1.8
14.0 0.6

20.9 2.2
18.2 1.1
9.4 1.9
10.9 1.1

Values are averages of triplicate analysis standard error.

the other extraction methods. Therefore, HRE was selected as the

optimal extraction method for TEAS. HRE extraction parameters
including ethanol concentration, extraction temperature, extraction time, solid-to-solvent ratio and extraction number were subsequently screened to determine the most inuential factors.
Although the experiments demonstrated that the solid-to-solvent
ratio and extraction number had relatively little effects on the
TPC, the result came out to be slightly higher when the solid-tosolvent ratio was 1:50. Therefore, the ratio was set at 1:50 (w/v)
and applied identically in all extractions.
The RSM allowed determining response TPC under the 20 sets of
conditions in TEAS. The response variable (Y) as follow
respectively;
Y1 = 69.01 + 3.85X1 + 1.07X2 + 4.86X3  10.32X21  9.52X22
 2.92X23 + 2.22X1X2 + 0.11X1X3  3.83X2X3
Y2 = 53.84 + 1.64X1 + 0.96X2 + 3.80X3  6.50X21  7.34X22
 4.12X23 + 2.09X1X2  0.07X1X3  3.06X2X3
Y3 = 29.21 + 1.66X1 + 0.46X2 + 2.33X3  3.53X21  3.56X22
 3.73X23 + 0.56X1X2 + 0.18X1X3  1.23X2X3
Y4 = 36.80 + 2.32X1 + 0.52X2 + 2.69X3  6.01X21  2.71X22
 5.03X23 + 0.72X1X2 + 0.50X1X3  1.56X2X3
Y1: green tea, Y2: oolong tea, Y3: black tea, Y4: mate.
In this study, the adequacy of the obtained model was determined by ANOVA test. Joglekar and May (Joglekar & May, 1987)
suggested that for a good t of a model, R2 should be at least
0.80. The ANOVA results conrmed a good model with R2 and R2
adjusted in the ranges of 0.930.96 and 0.880.93, respectively.
To aid visualization, the 3D response surface of the TPC, which
was generated during the extracting process of TEAS is shown in
Fig. 1. The optimum regions are where maximum TPC values can
be achieved, and those predicted TPC values and optimum extraction conditions are as follow: TPC value was 71.50 mg at the
extraction time of 123 min, extraction temperature of 70 C, and
ethanol concentration of 75% for green tea. The value was
54.82 mg at the extraction time of 98 min, extraction temperature
of 70 C, and ethanol concentration of 69% for oolong tea. And it
was 29.78 mg TPC at the extraction time of 105 min, extraction
temperature of 71 C, and ethanol concentration of 63% for black
tea. For mate, TPC value was obtained as 37.41 mg at the extraction
time of 103 min, extraction temperature of 71 C, and ethanol concentration of 61% for mate.
3.2. Method validation
The developed RP-HPLC/UV method was validated to verify that
its performance was compatible with the required performance for
the routine analysis of phenolic compounds and caffeine from
TEAS. The performance characteristics taken into account for
validation of the measurement method were linearity, sensitivity,
selectivity and accuracy, and precision.

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I.K. Bae et al. / Food Chemistry 172 (2015) 469475

Fig. 1. 3D response surfaces contour plots showing the effects of different extraction parameters (extraction time (X1), extraction temperature (X2), ethanol concentration
(X3)) on the response (TPC) in TEAS.

The linearity was evaluated by building external calibration

curves for each compound using a working solution containing
the 15 phenolic compounds and caffeine. Calibration curves were
obtained by plotting the analyte peak area versus its concentration
for seven different concentrations. Each concentration of the mixed
standard solution was injected in triplicate and regression parameters were calculated. These results demonstrated that external
standard calibration could be applied for quantitative purposes.
Coefcients of correlation (r2) better than 0.999 were achieved
for all compounds studied and they were shown in Table 3 with
detailed data of the regression equation.
The sensitivity of the developed method was assessed by determining the limits of detection (LOD) and quantication (LOQ). The
LOD and LOQ under the present chromatographic conditions were
calculated on the basis of the response and slope of each regression
equation at signal-to-noise ratios (S/N) of 3:1 and 10:1, respectively. For different components, the LOD values ranged from
0.06 mg/L to 2.92 mg/L, while the LOQ values ranged from
0.21 mg/L to 9.72 mg/L. Related data are also presented in Table 3.
The selectivity was evaluated by the absence of interference in
the same chromatographic windows as examined in the mixed

standard solution and analysis of the method blank (extraction solvent). No interfering peaks were observed in the blank chromatograms at the quantication wavelengths (270 and 320 nm).
Besides, as seen in Fig. 2 and Table 3, separation of all 16 compounds was successfully achieved in less than 32 min with good
resolution obtaining narrow and symmetric peaks. These results,
which identication of peaks were derived from the 16 studied
compounds, showed satisfactory selectivity on the HPLC system.
The precision of the developed method was determined by
measuring intra- and inter-day precisions. For intra-day precision,
the mixed standards solutions were analysed for six replicates
within 1 day, while for inter-day precision, the solutions were
examined in triplicates for consecutive 3 days. The precision was
expressed as the percentage of relative standard deviations
(% RSD). The overall% RSD values for both intra- and inter-days
were less than 4.3%, which indicates that the proposed method is
accurate (Table 3).
The accuracy was evaluated by adding the mixed standard
solutions with two different concentration levels (high and low)
to the known amounts of TEAS. Then, the mixtures were extracted
and analysed using the developed HRE and HPLC method. Six

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I.K. Bae et al. / Food Chemistry 172 (2015) 469475

Table 3
Validation parameters of the developed RP-HPLC/UV method.
Compound

Gallic acid
Gallocatechin
Epigallocatechin
Caffeine
3-Hydroxybenzoic acid
Caffeic acid
Chlorogenic acid
Epicatechin
Epigallocatechin
gallate
Gallocatechin gallate
Ferulic acid
Sinapic acid
Rutin
Epicatechin gallate
Catechin gallate
Quercetrin
a
b
c
d
e
f
g
h

Linear range
(mg/L)

Intercept SEa

0.5814.50
5.15128.75
9.72243.00
0.215.25
3.0075.00
0.7919.75
1.5639.00
1.1328.25
8.14203.50

53,100 22,302
30,500 6100
42,900 5148
160,000 64,000
170,000 35,700
218,000 39,240
154,000 92,400
120,000 106,800
297,000 59,400

9.47236.75
0.205.00
0.266.50
0.6215.50
1.2431.00
2.7669.00
0.338.25

25,600 6400
237,000 94,800
307,000 184,200
3940 3349
723,000 325,350
178,000 110,360
71,200 28,480

Slope SE

42,400 339.20
4470 17.88
2990 2.99
41,600 457.6
6610 46.27
86,000 688
35,300 141.2
2560 7.68
12,500 25
8390 8.39
103,000 206
90,100 630.7
14,700 132.3
4860 19.44
26,800 80.4
37,200 74.4

r2b

LODc
(mg/L)

LOQd
(mg/L)

Rse

Asymm.f

Precisions
(% RSDg)

Recovery
(%)h (n = 6)

Intraday
(n = 6)

Interday
(n = 9)

0.9998
0.9997
0.9998
0.9999
0.9999
0.9995
0.9997
0.9991
0.9996

0.18
1.55
2.92
0.06
0.90
0.24
0.47
0.34
2.44

0.58
5.15
9.72
0.21
3.00
0.79
1.56
1.13
8.14

14.5
17.2
2.2
7.4
3.8
3.4
2.1
14.7

0.95
1.04
1.01
0.94
0.95
0.91
1.03
0.92
1.39

1.4
3.8
1.7
4.3
2.9
3.2
4.2
2.1
0.7

0.8
1.8
3.4
2.6
2.1
1.4
2.8
4.2
0.9

98.4
101.2
99.0
103.5
101.7
96.6
99.4
98.1
96.9

0.9994
0.9996
0.9995
0.9998
0.9999
0.9996
0.9991

2.84
0.06
0.08
0.19
0.37
0.83
0.10

9.47
0.20
0.26
0.62
1.24
2.76
0.33

6.2
1.8
1.5
7.1
2.7
3.1
11.1

0.95
0.90
1.04
1.16
1.36
1.23
1.25

3.1
2.6
0.5
1.5
3.0
1.9
2.4

1.0
3.2
0.8
2.2
1.5
3.1
3.7

100.3
100.4
102.5
102.3
97.8
98.2
98.0

Standard error.
Coefcients of correlation.
Limit of detection, the lowest analyte concentration that produces a response detectable above the noise level of the system.
Limit of quantication, the lowest level of analyte that can be accurately and precisely measured.
Resolution.
Asymmetry.
Relative standard deviation, expressed as %.
Average of recoveries at two spike levels (high and low).

Fig. 2. HPLC chromatograms of a mixture of 15 phenolic compounds and caffeine detected at 270 and 320 nm. (1) gallic acid, (2) gallocatechin, (3) epigallocatechin,
(4) caffeine, (5) 3-hydroxybenzoic acid, (6) caffeic acid, (7) chlorogenic acid, (8) epicatechin, (9) epigallocatechin gallate, (10) gallocatechin gallate, (11) ferulic acid, (12)
sinapic acid, (13) rutin, (14) epicatechin gallate, (15) catechin gallate and (16) quercetrin.

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Table 4
Determination of the contents (mg/g) of 16 compounds in TEAS using the proposed method.
Peak#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
a

Compound
Gallic acid
Gallocatechin
Epigallocatechin
Caffeine
3-Hydroxybenzoic acid
Caffeic acid
Chlorogenic acid
Epicatechin
Epigallocatechin gallate
Gallocatechin gallate
Ferulic acid
Sinapic acid
Rutin
Epicatechin gallate
Catechin gallate
Quercetrin

Green tea
4.97 1.7
7.05 3.3
6.00 4.0
20.50 1.2

0.79 3.5
5.48 1.4
11.04 0.7
17.34 3.6

0.67 2.9
0.47 2.3
1.80 0.8

Oolong tea

Black tea

Mate

5.12 1.1
3.79 1.3
2.90 0.8
22.21 2.4

0.39 0.1
0.80 0.2

1.49 0.8

0.81 0.1

6.55 2.1

20.09 2.4

0.95 0.3
1.47 0.5

2.18 0.4

11.30 1.3

0.76 0.2
22.32 1.0

1.36 0.2
1.87 0.3
10.92 1.1

Values are averages of triplicate analysis standard error.

replicates were performed for the test. The percentage recoveries

were calculated according to the following equation: (total
detected amount  original amount)/added amount  100%. As a
result of calculation, the recovery rates were obtained within the
range of 96.6103.5% with RSD less than 3.9%, and that indicates
that the method has good accuracy (Table 3).
The validation data showed that the proposed method provides
good linearity, sensitivity, selectivity, accuracy as well as precision
for the simultaneous analysis of 15 phenolic compounds and caffeine in TEAS.
3.3. Applications of the optimized method
The developed method of HRE with RSM and RP-HPLC/UV was
applied to determine the contents of the 15 phenolic compounds
and caffeine from the TEAS (Table 4). Each sample was analysed
in triplicate. Identications of the 16 compounds were based on
their retention times in comparison with standards, as well as
the UV spectra of individual peaks in comparison with pure compounds. The qualitative and quantitative compositions of 16 compounds in the TEAS was varying signicantly. Among the analysed
TEAS, green tea had the highest content (49.18 mg/g) of catechins,
while oolong and black teas contained less (8.99 and 1.48 mg/g,
respectively). The reason for the differences of catechins content
in teas is due to the oxidation and polymerization of catechins
by enzymes derived from tea leaves during the production of
oolong and black tea from green tea by fermentation (Nishitani &
Sagesaka, 2004). Caffeine was found as the highest content in TEAS.
Mate contained no catechins, and to be compared to other teas, it
had much lower caffeine content and signicantly higher contents
of chlorogenic acid and rutin. The results obtained from mate are
correspond to those reported in the literature for simultaneous
analysis of the phenolic compounds and caffeine in different beverages (Rostagno et al., 2011).
4. Conclusion
A simple qualitative and quantitative method for simultaneous
determination of 15 phenolic compounds and caffeine from TEAS
was successfully developed and validated using RP-HPLC/UV
detection. Additionally, the extraction process was optimized using
RSM based on a CCD and resulted in the best conditions (extraction
time, 123, 98, 105, and 103 min; extraction temperature, 70, 70, 71,
and 71 C; and ethanol concentration, 75%, 69%, 63%, and 61%) for
the HER of phenolic compounds form TEAS. The proposed method

showed appropriate accuracy and precision, and was successfully

used for analysing different types of TEAS. The satisfactory results
demonstrated that the HPLC method provides a good alternative
for routine analysis owing to its simplicity, specicity and sensitivity and the potential to be applied as a reliable quality evaluation
method for TEAS.
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