Food Chemistry 171 (2015) 405411

Contents lists available at ScienceDirect

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

Analytical Methods

Chemical ngerprint analysis for quality control and identication

of Ziyang green tea by HPLC
Xiaoye He a, Jianke Li a,, Wei Zhao a, Run Liu a, Lin Zhang a, Xianghong Kong b
a
b

College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xian, Shaanxi 710062, PR China
Entry-Exit Inspection and Quarantine Bureau of Shaanxi, Xian, Shaanxi 710068, PR China

a r t i c l e

i n f o

Article history:
Received 28 March 2014
Received in revised form 1 September 2014
Accepted 6 September 2014
Available online 16 September 2014
Chemical compounds studied in this article:
Epigallocatechin gallate (PubChem CID:
65064)
Epigallocatechin (PubChem CID: 72277)
Epicatechin gallate (PubChem CID: 107905)
Epicatechin (PubChem CID: 72276)
Gallocatechin gallate (PubChem CID:
199472)
Gallocatechin (PubChem CID: 9882981)
Catechin (PubChem CID: 9064)
Gallic acid (PubChem CID: 370)
Chlorogenic acid (PubChem CID: 1794427)
Caffeine (PubChem CID: 2519)

a b s t r a c t
A simple and reliable HPLC ngerprint method was developed and validated for the quality control and
identication of Ziyang green tea. Ten batches of Ziyang green tea collected from different plantations in
Shaanxi Ziyang of China were used to establish the ngerprint. The feasibility and advantages of the used
HPLC ngerprint were veried for its similarity evaluation by systematically comparing chromatograms
with professional analytical software recommended by State Food and Drug Administration (SFDA) of
China. The similarities of the ngerprints of 10 batches of tea samples were all more than 0.981. Additionally, simultaneous quantication of 10 major bioactive ingredients in the tea samples was conducted to
interpret the consistency of the quality test. The results indicated that the HPLC ngerprint as a
characteristic distinguishing method combining similarity evaluation and quantication analysis can
be successfully used to assess the quality and to identify the authenticity of Ziyang green tea.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Ziyang green tea
Chemical ngerprint
HPLC
Similarity analysis
Quantitative determination

1. Introduction
Tea is the most widely consumed beverage in the world which
ranks after water (Wang et al., 2013) and is especially popular in
Asian countries (Lee et al., 2006). The chemical composition of
tea is complex and includes polyphenols, alkaloids (caffeine, theophylline and theobromine), amino acids, carbohydrates, proteins,
chlorophyll, volatile compounds, minerals, trace elements and
other unidentied compounds (Karori, Wachira, Wanyoko, &
Ngure, 2007). Among these compositions, polyphenols constitute
the most interesting group and are the main bioactive molecules
found in tea (Cabrera, Gimnez, & Lpez, 2003). The major
polyphenolic compounds in tea are avan-3-ols (i.e. catechins)
Corresponding author. Tel.: +86 29 85310519; fax: +86 29 85310517.
E-mail address: jiankel@snnu.edu.cn (J. Li).
http://dx.doi.org/10.1016/j.foodchem.2014.09.026
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

which include: (+)-catechin (C), ( )-epicatechin (EC), ( )-epigallocatechin (EGC), ( )-epigallocatechin gallate (EGCG), ( )-gallocatechins (GC), ( )-epicatechin gallate (ECG), and ( )-gallocatechin
gallate (GCG) (Kerio, Wachira, Wanyoko, & Rotich, 2013). The tea
beverage has been continuously considered as a medicine since
the ancient times because of its polyphenols. Numerous studies
have recorded the benecial effects of tea, which include antioxidant (Vinson & Dabbagh, 1998), anti-carcinoma (Sadzuka,
Sugiyama, & Sonobe, 2000), anti-inammation (Karori, Ngure,
Wachira, Wanyoko, & Mwangi, 2008), and antimicrobial properties
(Vaquero, Alberto, & Maca de Nandra, 2007). Therefore, tea appears
to be an effective chemopreventive agent for toxic chemicals and
carcinogens.
Over the last decade, consumers have shown strong interest in
foods with identied place of origin (Luykx & van Ruth, 2008). This
has brought more and more products with geographical

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X. He et al. / Food Chemistry 171 (2015) 405411

indications to the market. Therefore, the determination of geographical origin has become increasingly essential (Cordella,
Moussa, Martel, Sbirrazzuoli, & Louisette, 2002; Luykx & van
Ruth, 2008). One geographically specic selenium-enriched Ziyang
tea (C. sinensis L.) is widely distributed in the seleniferous region, in
Ziyang County, Shaanxi province, China (Wang, Zhao, Sun, & Yang,
2014). The selenium-enriched tea is currently being explored as
one promising dietary selenium supplement (Fang, Wu, & Hu,
2003). Interestingly, selenium-enriched green tea is shown to exhibit signicantly higher antioxidant activity than regular green tea
via in vitro experiments (Li et al., 2008).
In the event of frauds or commercial disputes, it is necessary to
introduce sensitive, accurate and fast detection methods as the
supplement of the food traceability system to prevent and verify
the fake information and products (Zhang, Zhang, Dediu, &
Victor, 2011). Chromatographic ngerprint, a comprehensive and
quantiable identication method, is able to reveal chemical information of botanical products, and therefore could be applied in
food adulteration detection (Ma et al., 2011). It has the potential
to characterise both the marker components and the unknown
components in a complex system (Li et al., 2010). Both U.S. Food
and Drug Administration (FDA) (FDA, 2004) and European Medicines Agency (EMEA) (EMEA, 2006) have recommended this strategy to assess the quality and consistency of botanical products.
State Food and Drug Administration of China (SFDA) has also
required that all the injections made from herbal medicines be
standardised by chromatographic ngerprinting (SFDA, 2000).
Moreover, SFDA has also suggested that all of herbal chromatograms should be evaluated by their similarities, a commonly
employed approach based on calculating the correlative coefcient
of original data (Gong, Liang, Xie, & Chau, 2003). Among the chromatographic ngerprinting applied to the authentication and qualitative evaluation of botanical products over the past decade (Chen,
Qi, & Shi, 2013; Ding et al., 2011; Liu, Wu, Yang, Ding, & Wu, 2013;
Zhang, Cui, He, Yu, & Guo, 2005), high performance liquid chromatography (HPLC) ngerprint emerges to be the most widely used
method attributed to its convenience and efciency. Also HPLC is
by far the most acceptable method for the analysis of tea catechins,
gallic acid, purine alkaloids, theanine, etc. (Peng, Song, Shi, Li, & Ye,
2008).
According to the best of our knowledge, there are no reports
regarding the ngerprint analysis of Ziyang green tea. The objective of this study was to establish an effective HPLC ngerprint
method for the identication and quality evaluation of Ziyang
green tea. The chromatograms of the extracted samples from different tea plantations of Shaanxi Ziyang were compared visually
and analysed by similarity evaluation. Moreover, ten components
in ten batches of Ziyang green tea were simultaneously quantitated
by HPLC method.

2. Materials and methods

2.1. Chemicals and solutions
HPLC grade methanol and triuoroacetic acid (TFA,
purity P 99%) were obtained from Fisher Scientic International
Inc. (Fair Lawn, New Jersey, USA) and Tokyo Chemical Industry
Co., Ltd. (Shanghai, China), respectively. Analytical-reagent grade
anhydrous ethanol was obtained from Tianjin Fuyu chemical Co.,
Ltd. (Tianjin, China). Deionised water used for all the solutions
and dilutions was prepared by Ultrapure Water Polishing System
of Shanghai Moller scientic instrument Co., Ltd. (Shanghai, China).
Ten chemical standards were purchased from SigmaAldrich Co.
(St. Louis, Missouri, USA), which are ( )-epigallocatechin gallate
(EGCG), ( )-epigallocatechin (EGC), ( )-epicatechin gallate (ECG),

( )-epicatechin (EC), ( )-gallocatechin gallate (GCG), ( )-gallocatechin (GC), (+)-catechin (C), gallic acid (GA), chlorogenic acid
(CA), and caffeine (CAF). The purity of these standards was all
above 98%.
Stock solutions (2 mg/mL) of the ten marker compounds were
prepared in methanol and stored at 4 C in darkness for further
analysis. Standard solutions were prepared by serial dilution of
the stock solution to the mobile phase working range of each substance. The concentration of mixed standard solution was selected
according to the level of the components that are expected in the
tea samples. The standard solutions were ltered through
0.45 lm lters prior to HPLC analysis.
2.2. Apparatus and chromatographic conditions
Waters 1525 HPLC system (Waters Corp., Milford, Massachusetts, USA) consisted of a binary pump, an ultraviolet detector
(Waters 2487) and a column temperature controller. System control and data analysis were processed with Waters Empower 2
software. The chromatographic separation was performed on an
Agilent Zorbax SB-C18 column (5 lm, 4.6 mm  250 mm) using
methanol-0.1% TFA solution (solvent A; 5:95, v/v; pH 2.28) and
methanol (solvent B) as mobile phase at a ow rate of 1 mL/min.
The gradient programme was set as follows: 010 min, 019% B;
1020 min, 1922% B; 2030 min, 2228% B; 3035 min, 2835%
B; 3540 min, 3535% B; 4050 min, 3565% B; 5060 min, 65
0% B. The chromatogram was monitored at a wavelength of
278 nm during the experiment. The column temperature was
maintained at 30 C and the injection volume of each sample and
standard solution was 20 lL. The HPLC mobile phase was prepared
fresh daily, ltered through a 0.45 lm membrane lter and then
degassed before injecting.
2.3. Plant materials and sample preparation
Ten batches of fresh tea leaves were collected from different
areas of Ziyang in Shaanxi province in May, 2013. Among these
ten batches of fresh tea leaves, Samples 14 were collected from
four tea plantations in Hongchun Town, Sample 7 and 8 were
obtained from two tea plantations in Dongmu Town, Sample 5, 6,
9 and 10 were picked from tea plantations in Gaoqiao Town, Haoping Town, Shuanghe Town, and Huangu Town, respectively. The
breed of all the tea trees was Ziyang population, which was a large
group distributed most widely in Shaanxi province.
Through basic production process of making green tea (including de-enzyming, rolling, and drying), the dried green tea bud
leaves were prepared. Each of the dried samples was pulverised,
and then sifted through a 250 lm sieve. 1.0 g dried power was precisely weighed and then put into a 100 mL conical ask with cover.
Approximately 25 mL of 50% ethanol solution was added to the
ask. Followed by heating in 80 C water bath for 20 min, the
extracted solution was cooled to room temperature and was ltrated through an analytical lter paper. The ltered solution was
then vacuum-dried at 40 C and the dried extract was dissolved in
100 mL 35% methanol solution. The sample was ltrated through a
0.45 lm membrane lter prior to HPLC analysis.
2.4. Standard curves, limits of detection and recovery rates of
quantitative analysis
The standard curves were obtained by plotting the peak area
against nominal concentration of each compound and were tted
to a linear function of type y = ax + b. In this equation, y and x represent peak area and nominal concentration in mg/L, respectively.
The limit of detection (LOD) was estimated as the minimum concentration of the compounds needed to produce signals that were

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X. He et al. / Food Chemistry 171 (2015) 405411

at least three times stronger than the noise signal (S/N P 3). The
accuracy tests were carried out by spiking the known contents of
mixed standard solution into the known concentration of Ziyang
green tea samples, and the assessment was done by analysing
the three different spiking concentrations of analytes in triplicates.
The percent recovery rates for the analytes were presented as
mean (100%).

HPLC-UV of ten target compounds in Ziyang green tea and reference standards, maximum absorbance values around 232 nm and
278 nm were observed. More detectable peaks could be obtained
and the baseline was well improved around 278 nm, and therefore
better characterisation of polyphenols could be achieved. Hence
characteristic chromatographic patterns were obtained by using
278 nm as the detection wavelength. Optimal HPLC condition used
in this study was shown in Section 2.2.

2.5. Method validation of HPLC ngerprint analysis

The precision of the HPLC ngerprint method was determined
by analysing the replicated extraction solution of the same sample
for ve times within a day. The sample stability test was determined with one sample on two consecutive days. The repeatability
was assessed by analysing ve independently prepared extraction
solutions of Ziyang green tea samples. During this period, the solution was stored at room temperature.

3.2. Method validation of quantitative analysis

The method of quantitative analysis was validated in terms of
linearity, limit of detection (LOD) and recovery test. Linear regression analysis for each compound was performed by plotting the
peak area (y) against the concentrations (x, mg/L) of the mixed
Table 1
Recovery rates of the ten components in Ziyang green tea (Sample 1) (n = 3).

2.6. Establishment of HPLC ngerprint and similarity analysis

Component

To establish the representative chromatographic ngerprint,

ten batches of Ziyang green tea samples were analysed under the
established HPLC method. The obtained HPLC data from ten
batches of Ziyang green tea samples were exported from Waters
Empower 2 software in AIA format and imported to the professional software named Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A)
(Ma et al., 2011). This system could reect the similarity of distribution ratio of the chemical composition accurately, which was
recommended by SFDA.

GA

3. Results and discussion

Original
(mg/L)

Added
(mg/L)

Found
(mg/L)

Recovery
rate (%)

RSD
(%)

1.90

0.50
1.00
2.00

2.39
2.79
3.81

99.72
96.32
97.78

1.74
2.33
1.58

GC

4.40

1.00
2.00
4.00

5.28
6.54
8.68

97.22
102.24
103.33

1.23
0.93
2.24

EGC

32.68

7.50
15.00
30.00

38.79
49.49
61.08

96.55
103.79
97.45

1.27
1.11
0.90

3.34

0.75
1.50
3.00

4.04
4.69
6.36

98.70
96.83
100.26

1.83
2.56
2.05

CA

3.22

0.75
1.50
3.00

4.00
4.52
5.94

100.67
95.83
95.44

2.16
2.80
0.93

234.69

50.00
100.00
200.00

291.86
326.29
416.28

102.52
97.49
95.76

2.07
1.83
2.11

CAF

77.53

17.50
35.00
70.00

96.04
108.37
142.41

101.06
96.31
96.53

0.77
2.13
1.03

EC

10.73

2.50
5.00
10.00

13.21
16.38
20.58

99.85
104.13
99.26

2.17
1.40
2.68

GCG

4.58

1.00
2.00
4.00

5.67
6.41
8.15

101.61
97.47
94.99

1.68
2.74
1.61

ECG

41.25

10.00
20.00
40.00

50.95
60.79
76.82

99.41
99.24
94.54

1.58
1.88
2.35

3.1. Optimisation of HPLC chromatographic conditions

EGCG

In order to obtain the most useful chemical information and

best separation in the ngerprint chromatograms of Ziyang green
tea, the mobile phase compositions, gradient elution procedure
and detection wavelength were optimised. In order to enhance
the resolution as well as to restrain the ionisation of target compounds, glacial acetic acid and TFA were added to the binary mixture of methanolwater. As a result, mobile phases containing TFA
were selected. To acquire better selectivity and higher efciency,
different concentrations of TFA (0.05%, 0.1% and 0.5%) in the methanol phase were also investigate. At the end, the mobile phase consisting of methanol and methanol-0.1% TFA solution (5:95, v/v, pH
2.28) was chosen for the determination of Ziyang green tea with
large number of peaks on the chromatogram achieving within
60 min. On the ultraviolet spectra with chromatograms of

Table 2
Analytical results of precision, stability and repeatability of 13 characteristic common peaks in Ziyang green tea samples (Sample 1) (n = 5).
Peak No.

1
2
3
4
5
6 (S)
7
8
9
10
11
12
13

RSD of RRT (%)

RSD of RPA (%)

Precision

Stability

Repeatability

Precision

Stability

Repeatability

0.19
0.33
0.24
0.20
0.20
0.00
0.19
0.08
0.13
0.09
0.15
0.15
0.16

0.18
0.11
0.14
0.12
0.11
0.00
0.08
0.03
0.08
0.14
0.24
0.29
0.26

0.15
0.20
0.05
0.18
0.09
0.00
0.21
0.07
0.11
0.17
0.15
0.16
0.31

2.32
0.90
1.24
1.60
1.19
0.00
1.77
2.19
1.69
2.92
0.47
0.78
0.93

1.32
1.99
1.50
1.60
1.67
0.00
1.67
2.01
1.31
1.64
1.13
1.03
1.00

2.50
2.31
1.05
1.83
1.09
0.00
1.67
1.69
0.94
2.87
1.36
1.54
2.19

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X. He et al. / Food Chemistry 171 (2015) 405411

standard
solutions
which
was
expressed
as
follow:
y = 79228x + 14,421, r2 = 0.9994 (for GA, the linear range is 0.5
10.0 mg/L); y = 5328.9x + 2632.6, r2 = 0.9990 (for GC, the linear
range is 2.020.0 mg/L); y = 5257.7x + 29,637, r2 = 0.9991 (for
EGC, the linear range is 20.0200.0 mg/L); y = 20915x 2080.2,
r2 = 0.9996 (for C, the linear range is 1.010.0 mg/L);
y = 23760x 2284.1, r2 = 0.9995 (for CA, the linear range is 0.5
10.0 mg/L); y = 32815x + 29,836, r2 = 0.9995 (for EGCG, the linear
range is 40.0400.0 mg/L); y = 69685x + 95,859, r2 = 0.9998 (for
CAF, the linear range is 20.0200.0 mg/L); y = 17811x + 26,182,
r2 = 0.9991 (for EC, the linear range is 5.050.0 mg/L);
y = 35274x 2135.2, r2 = 0.9997 (for GCG, the linear range is 0.5
20.0 mg/L); y = 51003x + 101,605, r2 = 0.9996 (for ECG, the linear

range is 20.0200.0 mg/L). All the standard curves showed good

linear regression (R2 P 0.9990) within test ranges.
The LOD under the present HPLC method were determined at
signalnoise ratio (S/N) of 3. Standard solution containing ten reference compounds was diluted to a series of appropriate concentrations with methanol. The diluted solutions were injected into
HPLC for analysis. The LOD for GA, GC, EGC, C, CA, EGCG, CAF, EC,
GCG, and ECG were 0.018, 0.153, 0.126, 0.039, 0.043, 0.034,
0.028, 0.064, 0.038, and 0.026 mg/L, respectively, which indicated
that the analytical method was acceptable with sufcient
sensitivity.
The recovery test was determined by standard addition method.
The samples (Sample 1) were spiked with the high, intermediate

(A)

Gallic acid (GA)

Chlorogenic acid (CA)

Catechin (C)

Epicatechin (EC)

Epicatechin gallate (ECG)

Caeine (CAF)

Gallocatechin (GC)

Gallocatechin gallate (GCG)

Epigallocatechin (EGC)

Epigallocatechin gallate (EGCG)

(B)
0.40

0.35

10

0.30

AU

0.25

0.20

0.15

0.10

0.05
0.00
0.00

2.00

4.00

6.00

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

tR (min)
Fig. 1. The chemical structures of the investigated compounds (A) and the typical HPLC chromatographic prole of ten standards (B). HPLC peaks: 1, GA (8.032 min); 2, GC
(10.398 min); 3, EGC (15.013 min); 4, C (15.521 min); 5, CA (18.290 min); 6, EGCG (20.165 min); 7, CAF (21.056 min); 8, EC (23.148 min); 9, GCG (25.683 min); 10, ECG
(31.218 min).

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X. He et al. / Food Chemistry 171 (2015) 405411

and low levels of mixed standard solution of ten components in

triplicates, and then extracted, processed and quantied in accordance with the established procedures (Section 2.2). The results
of the recovery rates are summarised in Table 1. The recovery rates
of the ten compounds were in the range of 94.54104.13% and
their RSD values were less than 2.80%. These validation results
indicated that the conditions used in the quantitative determination were appropriate.

calculated by analysing ve independently prepared solutions of

the same sample (Sample 1). The variation of RRT and RPA of the
characteristic peaks did not exceed 1% and 3%, respectively. The
intraday precision variation of the RRT and RPA of the characteristic peaks was below 1% and 3%, respectively, by analysing the ve
replicate sample solutions (Sample 1) continuously on the same
day. The interday stability test was assessed by analysing the same
sample solution (Sample 1) on two consecutive days at different
time intervals (0, 6, 12, 24, and 48 h) and the RSD values of RRT
as well as RPA of the characteristic common peaks were less than
1% and 3%, respectively. The observed results indicated that the
sample solution was stable within 48 h. The results of the precision, stability and repeatability are shown in Table 2, which met
the national standard of traditional Chinese medicine ngerprint
(SFDA, 2000).

3.3. Method validation of HPLC ngerprint analysis

The method of HPLC ngerprint analysis was validated in terms
of precision, repeatability and stability test. Intraday precision and
repeatability as well as interday stability of the HPLC ngerprint
method were determined and expressed by the relative standard
deviations (RSD) value of the average relative retention times
(RRT) and relative peak areas (RPA) of the 13 characteristic common peaks with the respect to the reference peak (peak 6) at retention time (tR) of 19.933 min. By using the optimised conditions
described above, the repeatability of the HPLC method was

3.4. HPLC ngerprints of Ziyang green tea samples

Using the optimised HPLC method, the peaks of ten investigated
chemical markers showed satisfactory resolution (Fig. 1B). Under

(A)

(B)
6

0.50

0.40

0.30

AU

0.20

12
4

0.10

11

13

9 10

0.00
0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

t R (min)
Fig. 2. HPLC chromatographic ngerprints of 10 Ziyang green tea samples (A) and the reference ngerprint of Ziyang green tea (B). (B): 1, GA (7.936 min); 2, Unknown 1
(8.540 min); 3, Unknown 2 (12.196 min); 4, Unknown 3 (13.831 min); 5, EGC (14.930 min); 6, EGCG (19.933 min); 7, CAF (20.890 min); 8, EC (22.923 min); 9, GCG
(25.425 min); 10, Unknown 4 (26.046 min); 11, Unknown 5 (28.864 min); 12, ECG (30.991 min); 13, Unknown 6 (46.880 min).

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X. He et al. / Food Chemistry 171 (2015) 405411

the optimal conditions, the HPLC ngerprints of ten batches of Ziyang green tea from different areas of Shaanxi Ziyang were
obtained, and were selected and matched by the Similarity
Evaluation System for Chromatographic Fingerprint of Traditional
Chinese Medicine (Version 2004A) (Fig. 2A). These samples had similar HPLC proles.
Peaks existed in the standard ngerprint with relatively high
intensity and good resolutions were assigned as characteristic
common peaks to represent the characteristics of samples. There
were 13 characteristic peaks (from peak 1 to 13) found in the chromatogram, which covered more than 90% of the total area (Fig. 2B).
Seven components were identied as GA (peak 1), EGC (peak 5),
EGCG (peak 6), CAF (peak 7), EC (peak 8), ECG (peak 9), and GCG
(peak 12) by comparing their retention time and UV spectrum with
those of standard compounds. The other six common ngerprint
peaks were unknown. To calculate the RRT and RPA of each characteristic peak, a reference peak should be chosen. Peak 6 (EGCG)
had a considerably high content of more than 30% of the total area,
and it also had moderate retention time, stable peak area and good
shape in the Ziyang green tea chromatograms. Therefore, it was
chosen as the reference peak (S). Then the retention time and peak
area of the 13 common peaks were measured, RRT and RPA of all
characteristic common peaks with respect to this reference peak
were calculated (Table 3).
Among the 13 characteristic common peaks, ve peaks RSD
values of RPA were under 25%, and these ve peaks accounted
for 77% of the total peak area, which indicated that they were
the main components in the Ziyang green tea samples. The largest
variation was found in the other eight peaks with all having an RSD

over 25% in RPA for the ten batches of samples. Considering the
individual differences in Ziyang green tea collected from different
regions, the concentrations of some ingredients are different from
batch to batch, especially those ingredients with low content in
green tea. The single peak area of the eight peaks was less than
10% of the total area, thus the RSD of RPA of the eight peaks could
not be monitored, however the RRT must be controlled in order to
guarantee the internal quality. The RSD of RRT of 13 characteristic
common peaks in the ten batches of samples was all under 1%,
which met the national standard.
3.5. Similarity analysis of HPLC ngerprints of Ziyang green tea
samples
The similarities between the entire chromatographic proles of
ten batches of Ziyang green tea and the standard chromatographic
ngerprint were calculated by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version
2004A), and the correlation coefcients of all samples ngerprints
were shown as 0.993, 0.998, 0.994, 0.997, 0.999, 0.981, 0.997,
0.998, 0.998, and 0.999. These results showed that the ten batches
of Ziyang green tea from different tea plantations shared nearly the
same correlation coefcients of similarities, which indicated that
the change of soil condition didnt inuence the quality of the sample. In general, the common pattern of the ten batches test samples
could be applied as a reference HPLC ngerprint to identify as well
as to assess Ziyang green tea.
3.6. Quantitative determination of ten components in Ziyang green tea

Table 3
The retention time (tR), relative retention time (RRT), peak area (PA) and relative peak
area (RPA) of 13 characteristic peaks in Ziyang green teas (n = 10).
Component

1
2
3
4
5
6 (S)
7
8
9
10
11
12
13

tR (min)

7.936
8.540
12.196
13.831
14.930
19.933
20.890
22.923
25.425
26.046
28.864
30.991
46.880

RRT

PA (mV s)

Average

RSD (%)

0.398
0.428
0.612
0.694
0.749
1.000
1.048
1.150
1.276
1.307
1.448
1.555
2.352

0.30
0.23
0.11
0.08
0.09
0.00
0.11
0.09
0.09
0.10
0.19
0.23
0.19

194,651
1,071,320
429,667
527,035
250,284
6,033,606
5,423,531
285,780
153,271
153,045
342,956
1,587,557
135,181

In this study, the proposed HPLC method was successfully

applied to the simultaneous determination of the ten components
in Ziyang green tea samples. The identity of the marker compound
peaks in the chromatogram was conrmed by their retention times
and their UV proles. Quantication was based on the external
standard method using calibration curves tted by linear regression analysis. The analysis was performed in triplicates. The contents of the ten marker compounds in ten batches of Ziyang
green tea from different areas of Ziyang are summarised in Table 4.
The results showed that the catechins (including EGCG, EGC,
ECG, EC, GCG, GC, and C) accounted for 77.66% of the contents of
ten compounds, which were the dominant components in Ziyang
green tea samples. Furthermore, EGCG was the major catechin
monomer found in the tea samples in the range of 48.68
93.88 mg/g, followed by EGC (average content was 19.04 mg/g),
ECG (12.80 mg/g), EC (6.66 mg/g), GC (2.86 mg/g), GCG (2.11 mg/g),
and C (1.44 mg/g). The identied epicatechins (EGCG, EGC, ECG
and EC) are in cis structure. The non-epicatechins GCG, GC and C
are the corresponding epimers of EGCG, EGC and EC, respectively

RPA
Average

RSD (%)

0.035
0.180
0.069
0.086
0.043
1.000
0.923
0.049
0.027
0.026
0.055
0.268
0.024

58.97
18.26
56.67
30.99
38.38
0.00
18.55
24.50
67.43
46.87
53.10
14.74
56.43

a
Component: 1, GA; 2, Unknown 1; 3, Unknown 2; 4, Unknown 3; 5, EGC; 6, EGCG;
7, CAF; 8, EC; 9, GCG; 10, Unknown 4; 11, Unknown 5; 12, ECG; 13, Unknown 6.

Table 4
Contents of ten components in Ziyang green teas (n = 3, mg/g).
Sample No.

S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
Average
RSD (%)

Content of investigated components

GA

GC

EGC

CA

EGCG

CAF

EC

GCG

ECG

0.76
1.11
0.71
0.38
1.25
1.62
1.54
0.88
1.26
1.31
1.08
36.29

1.76
4.27
1.72
1.97
4.48
2.95
5.15
2.44
2.04
1.85
2.86
45.15

13.07
23.37
12.26
26.87
24.96
14.05
18.03
21.58
18.49
17.68
19.04
26.63

1.33
1.46
1.20
1.32
1.70
1.18
1.52
1.48
1.69
1.56
1.44
12.74

1.29
0.33
0.43
0.19
0.62
0.39
0.57
0.70
0.72
1.91
0.72
72.22

93.88
61.10
62.32
75.89
66.96
48.68
66.05
79.91
65.26
85.45
70.55
18.77

31.01
26.71
32.15
29.28
27.60
29.60
32.95
34.27
32.52
38.06
31.42
10.67

4.29
6.65
6.12
6.14
6.48
7.69
7.04
8.12
5.42
8.61
6.66
19.32

1.83
3.34
0.76
0.71
3.65
1.86
5.75
1.28
0.86
1.05
2.11
78.12

16.50
10.99
13.00
11.25
12.25
12.20
11.03
13.05
12.76
14.92
12.80
13.73

X. He et al. / Food Chemistry 171 (2015) 405411

(Fig. 1A). The accumulation of EGCG in green tea is possibly due to

inactivation of the polyphenol oxidase by drying and steaming the
fresh leaves until non oxidation process occurs (Rahim, Nofrizal, &
Saad, 2014). Additionally, the phenolic acid compounds, including
GA (average content was 1.08 mg/g) and CA (0.72 mg/g), accounted
for 1.21% of the contents of ten markers. The level of caffeine in
Ziyang green tea samples was in the range of 26.7138.06 mg/g.
The largest variation was found in the contents of CA and GCG in
the ten batches of samples with having an RSD of 72.22% and
78.12%, respectively. This indicated that the contents of CA and
GCG in the ten batches of Ziyang green tea samples from different
tea plantations were evidently different. The other eight contents
in the ten batches of Ziyang green tea samples were consistent
with all having an RSD less than 50%. These data suggested that
the quality of the commercial Ziyang green tea could be successfully monitored by determining these contents in the samples.
4. Conclusion
In this study, a simple, accurate, and reliable method was developed to evaluate the quality of Ziyang green tea by using the established HPLC ngerprint and the determination of ten bioactive
compounds. The results showed that the method was reliable, with
high precision and good repeatability, and thus was suitable for the
quality assessment of Ziyang green tea samples. For the ngerprint
analysis, 13 characteristics ngerprint peaks were applied to evaluating the similarities among ten batches of Ziyang green tea and
showed good similarities. For the quantitative determination, ten
components of ten batches of Ziyang green tea were successfully
separated and determined. Considering its advantages of reliability
and sensitivity, the HPLC ngerprint method coupled with quantitative analysis has great potential to be widely used for identifying
the authenticity and assessing the quality of the Ziyang green tea.
Acknowledgements
This study was nancially supported by the National Natural
Science Foundation of China (No. 31171677), and Public Welfare
Special Program of State General Administration of the Peoples
Republic of China for Quality Supervision and Inspection and Quarantine (No. 201010040-2).
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