Food Chemistry 197 (2016) 1112–1120

Contents lists available at ScienceDirect

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

Determination and quality evaluation of green tea extracts through

qualitative and quantitative analysis of multi-components by single
marker (QAMS)
Da-Wei Li a, Ming Zhu b,⇑, Yun-Dong Shao c, Zhe Shen a, Chen-Chen Weng c, Wei-Dong Yan a,⇑
a
Department of Chemistry, Zhejiang University, Hangzhou 310027, China
b
Zhejiang Institute for Food and Drug Control, Hangzhou 310051, China
c
Skyherb Ingredients, Anji 313300, China

a r t i c l e i n f o a b s t r a c t

Article history: The quality of tea is mainly attributed to tea polyphenols and caffeine. In this paper, a new strategy for
Received 21 May 2015 quality evaluation of green tea extracts was explored and verified through qualitative and quantitative
Received in revised form 14 October 2015 analysis of multi-components by single marker (QAMS). Taguchi Design was introduced to evaluate
Accepted 19 November 2015
the fluctuations of the relative conversion factors (fx) of tea catechins, gallic acid and caffeine to epigal-
Available online 28 November 2015
locatechin gallate. The regression model (Sig. = 0.000) and the deviations (R2 > 0.999) between QAMS and
normal external standard method proved the consistency of the two methods. Hierarchical cluster anal-
Chemical compounds studied in this article:
ysis and canonical discriminant analysis were employed to classify 26 batches of commercial Longjing
Epigallocatechin gallate (PubChem CID:
65064)
green tea extracts (LJGTEs) collected from different producers. The results showed a significant difference
Epicatechingallate (PubChem CID: 367141) in component profile between the samples from different origins. The QAMS method was verified to be
Epigallocatechin (PubChem CID: 72277) an alternative and promising method to comprehensively and effectively control the quality of LJGTEs
Epicatechin (PubChem CID: 72276) from different origins.
Gallocatechin gallate (PubChem CID: Ó 2015 Elsevier Ltd. All rights reserved.
199472)
Gallocatechin (PubChem CID: 65084)
Catechin gallate (PubChem CID: 6419835)
Catechin (PubChem CID: 73160)
Caffeine (PubChem CID: 2519)
Gallic acid (PubChem CID: 370)

Keywords:
Determination
Quality evaluation
Qualitative and quantitative analysis of
multi-components by single marker (QAMS)
Green tea extracts
Taguchi Design
HPLC

1. Introduction
Green tea is one of the most popular and widely consumed bev-
erages all over the world. Phenolic compounds, the rich sources in
green tea extracts, are potentially beneficial for health, as they act
not only as strong antioxidant agents but also as many drug effica-
cies (Jankun, Selman, Swiercz, & Shrzypczak-Jankun, 1997; Yang,
⇑ Corresponding authors.
E-mail addresses: zhumingd@hotmail.com (M. Zhu), yanweidong@zju.edu.cn
(W.-D. Yan).
Wang, Lu, & Picinich, 2009; Zhao et al., 2014). They can be used
in the treatment of promoting blood circulation, improving liver
function and oral health, eliminating various toxins and inducing
apoptosis in human lymphoid leukemia cells (Fujiki et al., 1996;
Liao, Umekita, Guo, Kokontis, & Hiipakka, 1995; Stoner &
Mukhtar, 1995).
As the first rank of top ten teas in China, Longjing green tea has
been widely used in food, nutraceuticals, pharmaceutical products
and other areas. Tea polyphenols (TPs) were the most abundant
substances in Longjing green tea extracts (LJGTEs). It has been
reported that TPs was the primary contributor to affect the quality

http://dx.doi.org/10.1016/j.foodchem.2015.11.101
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
 D.-W. Li et al. / Food Chemistry 197 (2016) 1112–1120
of the tea in the flavor, color, aroma and healthy benefits (Zhao
et al., 2014). The catechins accounts for about 60–90% of TPs in
general. The catechins consists of epigallocatechin gallate (EGCG),
epicatechingallate (ECG), epigallocatechin (EGC), epicatechin (EC),
gallocatechin gallate (GCG), gallocatechin (GC), catechin gallate
(CG) and catechin (C) (Lee, Hwang, Lee, & Choung, 2014; vander
Hooft et al., 2012). Caffeine (CAF) and gallic acid (GA) in LJGTEs
were both somewhat harmful to human beings. Caffeine can trig-
ger palpitations and increase blood pressure in sensitive individu-
als. Gallic acid belongs to chemical raw materials. Because of the
possible adverse health effects, the content of them in the tea
extracts should be restricted. Chemical profiles greatly affect the
quality of green tea, which vary significantly among different ori-
gins (Maeda-Yamamoto, Ema, Monobe, Tokuda, & Tachibana,
2012; Serpen et al., 2012; Tian et al., 2012). Health claims need
to be backed by scientific study of the components, it is therefore
necessary to propose a method that could accurately determine
the components in LJGTEs.
In addition, the quality and benefits of LJGTEs were not solely
determined by the single compound, but rather the combinational
effects of multiple constituents (Yang, 1997). For the moment, a
large number of methods have been reported for the simultaneous
determination of multi-components (SDMC) in herbal or botanical
products. High performance liquid chromatography (HPLC) is
mainly used to determine the contents of most organic com-
pounds. Based on conventional HPLC, there were two main meth-
ods for SDMC. In the first method, multiple reference standards
were used for the analysis of multiple components, as the normal
external standard method (ESM). ESM has been most commonly
used for the qualitative and quantitative analysis of components
in herbal or botanical products and indeed has played an important
role in the fields of research, production regulation and evaluation
(Jin, Ma, Ma, Yao, & Chen, 2014; Samanidou, Tsagiannidis, &
Sarakatsianos, 2012; Wang & Helliwell, 2001; Zhang et al., 2013).
However, in the recent years, quality control and evaluation of Tra-
ditional Chinese Medicines (TCMs) by ESM has become challenging
in some cases due to the high cost of some reference standards, the
diversity and complexity of botanical products, the instability of
detection and many other uncertainties. According to some reports
(Gao, Jiang, Lu, & Tu, 2009; He, Li, Zhao, & Dong, 2012; He, Yang, &
Zhang, 2012; Hou et al., 2011), ESM may have relatively low speci-
ficity and low efficiency, and to some extent, it was challenging to
determine the chemical profile with ESM, which would make it dif-
ficult to evaluate TCMs comprehensively. It was especially difficult
to determine more than five components simultaneously in the
routine quality supervision and evaluation. Meanwhile, quality
control of botanical products (herb drugs or TCMs) by multi-
components has been acknowledged by both United States Phar-
macopeia (USP) and Chinese Pharmacopoeia (Ch.P.).
In comparison, the second method requires one single reference
standard to simultaneously determine the contents of multi-
components, which was abbreviated to QAMS (qualitative and
quantitative analysis of multi-components by single marker).
Qualitative and quantitative analysis of multi-components by sin-
gle marker can not only greatly reduce the detection time and the
cost of the experiment, but improve the practicability of the
method and control the quality of herbal or botanical products
more effectively and comprehensively. It has been reported that
QAMS has been applied in the determination of Rhubarb Rhizome
(Gao et al., 2009), Panax Ginseng (Li et al., 2013), Salvia Miltior-
rhiza (Hou et al., 2011) and other botanical ingredients (Du, Li,
Liu, Yin, & Bi, 2014; He et al., 2012; Wang, Gao, Fu, & Wang,
2006; Wang, Provan, & Helliwell, 2003). Furthermore, quality con-
trol of Cat’s Claw (United States Pharmacopeia, 2010a) and Bilberry
Extract (United States Pharmacopeia, 2010b) by QAMS have both
been included in USP 33-NF. In Ch.P. 2010 edition (volume I),
1113
simultaneous determination of multi-components in Coptides
Rhizoma (Huanglian) by QAMS had also been included (Chinese
Pharmacopoeia, 2010). It is yet to be studied that whether the rel-
ative conversion factor (fx) of QAMS in simultaneous determination
of multi-components in LJGTEs could be used as a constant in dif-
ferent laboratories, and what influenced fx (Wang et al., 2003;
Zhang et al., 2014). There is also a lack of study on whether QAMS
could be proposed to easily and effectively control the quality of
LJGTEs.
In our work, the new quality control of LJGTEs through QAMS
was presented for the simultaneous determination of eight tea cat-
echins, GA and CAF in LJGTEs. The applicability and feasibility of
QAMS mainly rely on the consistency of the environmental and
operational parametric variables, such as analysts, HPLC instru-
ments, and columns. In this study, the fluctuations and stability
of fx with a focus on different columns, HPLC instruments and ana-
lysts were evaluated and discussed (Goupy, 2005). The linear
regression model was built between two variables to examine
deviations of the content results obtained by QAMS and ESM.
Hierarchical cluster analysis (HCA) and canonical discriminant
analysis (CDA), which were based on the contents of eight
catechins were employed to classify 26 batches of LJGTEs collected
from different producers.
2. Materials and methods
2.1. Chemicals and materials
Eight catechins reference standards were purchased from
Chengdu Biopurity Phytochemicals Co., Ltd. The structures of these
standards were identified and confirmed through analyses with
UV, MS, 1H NMR and 13C NMR (see S1–S3 in Supporting Informa-
tion). CAF and GA were provided by National Institutes for Food
and Drug Control (NIFDC), which has Laboratory Accreditation Cer-
tificate. The purity of these ten compounds was verified to be
greater than 98% with HPLC using the peak area normalization
method. Methanol for HPLC and other related materials were
obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai,
China). Voucher specimens were stored at Zhejiang Institute for
Food and Drug Control. All 26 batches of LJGTEs were obtained
from seven major producers (Table 1).
2.2. Instruments and conditions
Analyses were primarily performed by a Wufeng HPLC-100
system composed of a double solvent delivery system, an on-line
degasser, a column temperature controller, and dual wavelength
UV detector coupled with an analytical workstation (WS-100
software) (Wufeng Tech. Shanghai, China). Analytes were separated
on a reverse phase C18 column (Thermo ODS hypersil column,
250 mm
4.6 mm
5 lm). Samples with a volume of 20 lL were
injected into the column and the column temperature was main-
tained at 40 °C. The mobile phase was programmed with a linear gra-
dient, 100% A (900:1:100, v/v; water, phosphoric acid, acetonitrile),
0% B (200:1:800, v/v; water, phosphoric acid, acetonitrile) for
10 min, from 0% B to 10% B in 20 min, from 10% B to 0% B in 20 min,
0% B for 5 min, at a flow rate of 1.0 mL/min. The system was
maintained at pre-equilibrium for 10 min between consecutive injec-
tions. The detection wavelength was set at 280 nm. Reproducibility
was verified by duplicate analyses. Two additional HPLC instruments
(see S4 in Supporting Information) and five representative additional
columns were used (see S5 in Supporting Information).
UV-TU1950 was from Beijing Purkinje General Instrument
Co., Ltd (Scanning range of 190–700 nm). 1H and 13C NMR spectra
were measured on a Bruker 400 MHz spectrometers with chemical
1114 D.-W. Li et al. / Food Chemistry 197 (2016) 1112–1120

Table 1
The content of ten compounds in 26 batches of investigated commercial LJGTEs samples (mg/g).

No. Producersa Batches GA GC EGC CAF C EC EGCG GCG ECG CG T1b T2c
s1 ZGP 0301a 17.1 34.5 181.7 24.6 12.9 59.2 449.4 13.2 100.0 10.2 861.1 902.8
s2 ZGP 1101a 16.4 24.5 155.6 21.7 3.2 57.8 441.1 12.8 113.9 8.4 817.3 855.4
s3 ZGP 0301b 10.6 38.5 183.5 23.8 9.6 62.6 476.0 15.6 106.8 10.5 903.1 937.5
s4 ZGP 0302 17.9 38.5 144.5 26.3 20.4 54 461.0 17.6 103.8 7.0 846.8 891
s5 ZGP 1002 10.8 38.7 170.4 26.6 20.8 55.8 505.3 20.5 125.8 7.5 944.8 982.2
s6 ZGP 1101b 13.5 31.6 174.2 24.1 8.1 61.5 475.3 15.7 107.2 7.0 880.6 918.2
s7 XGP 0801 23.0 24.2 128.5 2.8 2.3 51.2 464 16.2 106.9 7.4 800.7 826.5
s8 STP 1201 17.9 44.1 186.2 12.5 4.8 57.5 495.1 30.7 114.6 11.4 944.4 974.8
s9 STP 0401 16.4 37.7 182.4 12.9 3.9 54.6 524.6 25.5 114.1 4.6 947.4 976.7
s10 LWP 0301a 11.2 44.4 223.5 7.5 9.1 67.4 457.8 51.9 102.4 3.7 960.2 978.9
s11 HSP 1201 15.0 38.0 127.7 10.9 9.1 51.8 464.3 26.5 109.4 3.8 830.6 856.5
s12 ZGP 1227 14.3 38.9 112.5 7.1 9.3 57.3 430.1 22.7 99.9 4.4 775.1 796.5
s13 LWP 0101a 15.5 53.1 177.2 7.0 12.1 58.4 409.5 49.3 97.5 3.9 861 883.5
s14 SSP 0401 28.6 29.7 149.9 3.1 6.7 37.4 372.9 15.9 80.1 5.8 698.4 730.1
s15 SSP 0402 30.1 32.7 160.5 3.3 9.0 42.4 380.6 20.2 85.6 2.1 733.1 766.5
s16 SSP 0403 30.3 35.6 157.7 3.1 8.8 41.0 376.8 20.6 83.6 1.5 725.6 759.0
s17 SOP 0102 13.8 59.3 192.9 18.9 12.9 64.3 404.4 24.2 145.6 1.7 905.3 938.0
s18 SSP 0701 11.9 41.9 228 3.0 18.5 44.0 469.4 36.3 97.6 2.8 938.5 953.4
s19 SSP 0301 12.2 33.5 215.3 3.1 15.5 46.2 459.8 27.0 98.6 3.7 899.6 914.9
s20 SSP 1201 12.5 38.4 218.5 2.9 16.3 43.8 445.3 28.9 97 3.1 891.3 906.7
s21 SOP 0904 12.5 60.7 206.5 3.2 19.3 72.6 465.8 29.8 74.8 1.2 930.7 946.4
s22 LWP 0401b 11.0 40.3 226.8 7.4 11 62.3 450.7 51.5 98.5 1.2 942.3 960.7
s23 LWP 0401a 10.6 43.3 229.4 6.9 10.4 65.2 456.3 53.7 104.3 1.2 963.8 981.3
s24 LWP 0301b 16.2 55.8 225.6 7.5 6.7 62.8 418 58.6 103.8 4.9 936.2 959.9
s25 LWP 1101 14.2 56.5 194.3 6.2 5.9 63.9 412 50.2 99.3 4.3 886.4 906.8
s26 LWP 0101b 18.3 55.7 228.8 7.8 7.6 77.3 421.5 57.5 100.7 5.6 954.7 980.8
Minimum 10.6 24.2 112.5 2.8 2.3 37.4 372.9 12.8 74.8 1.2 698.4 730.1
Maximum 30.3 60.7 229.4 26.6 20.8 77.3 524.6 58.6 145.6 11.4 963.8 982.2
Means 16.2 41.2 183.9 10.9 10.5 56.6 445.9 30.5 102.8 5.0 876.1 903.3
Standard deviation 5.8 10.2 34.9 8.5 5.3 9.9 38.2 15.3 14.1 3.0 77.5 74.8
a
LJGTEs were collected from 7 major producers: HSP; SOP; SSP; LWP; ZGP; STP; XGP.
b
The total content of 8 catechins.
c
The total content of 10 compounds.

shifts reported as parts per million (in CD3OD at 20 °C). Mass
spectroscopy data of the compounds was collected on MS-ESI
(Esquire 3000plus) from Bruker Daltonics Inc.
2.3. Preparation of LJGTEs and sample solutions
The preparation of LJGTEs has been included in the national
drug standard (Prescription of Chinese Medicinal, 1998). Green
tea extracts were prepared by placing 10.0 g powder of Longjing
tea leaves into 50 mL boiled water and heated (100 °C) for
50 min. Then the solutions were first filtered, and another 50 mL
boiled water was added for second extraction. Then the saturated
limewater was gradually added in the combined filtrate until
pH = 9. Being equilibrated for 10 min, the solid precipitate was fil-
trated and then the hydrochloric acid solution (37%) was added to
dissolve the precipitate until pH = 4. Ethyl acetate was added to
extract the tea bioactive components from the solutions. The solu-
tions were evaporated under reduced pressure. Approximately 10–
15 mg tea extracts were dissolved with water/methanol (3:1, v/v)
10 mL, centrifuged at 10,000 rpm for 10 min prior to HPLC analysis.
2.4. Preparation of standard solutions
Stock solutions were prepared by dissolving approximately
10 mg of each reference standard respectively into a 10 mL volu-
metric flask with water/methanol (3:1, v/v) to volume, mixed,
and then diluted (dilution factor = 5) with water/methanol (3:1,
v/v) prior to HPLC analysis.
2.5. Calculation of relative conversion factors
In order to obtain fx for each analyte, it was essential to select a
suitable reference analyte. The selected reference analyte should
be abundant in each sample. It should be stable, easily obtainable
and have a clear pharmacological effect or related to clinical
efficacy. Moreover, it should be easily separated under the
conventional chromatographic conditions. Epigallocatechin gallate
(EGCG) was the most abundant and active component in LJGTEs
and it was used as a quality indicator in this paper for its low cost,
high stability, high content, easy to obtain and significant
pharmacological activities in LJGTEs.
The standard solutions were analyzed under the conditions
detailed above. Using EGCG as a reference standard, fx could be
calculated by the ratio of the peak areas and the ratio of the
concentration between the analyte and EGCG (Eq. (1)). With
the results of fx, the quantification of bioactive components (Cx)
in the samples could be carried out according to the following
equations (Eqs. (2) and (3)):
AEGCG =C EGCG
fx ¼ ð1Þ
Ax =C x
Ax
C EGCG
Cx ¼
fx ð2Þ
AEGCG
Cx
V
xx ¼ ð3Þ
m
where Ax is the peak area of the bioactive component in the single
standard solution or in the samples; fx is the average relative
conversion factor of each bioactive component to EGCG; Cx is the
concentration of analyte in the single standard solution or in the
samples (mg/mL); V is the volume of the green tea extracts
solutions (mL); m is the mass of the green tea extracts (mg); xx is
the mass concentration of the bioactive components in the green
tea extracts (mg/g)).
 D.-W. Li et al. / Food Chemistry 197 (2016) 1112–1120
The ESM method required that all the reference standards solu-
tions corresponding to analytes would be prepared first. Con-
versely, the QAMS method did not require preparation for all the
reference standards solutions instead only one standard solution
was needed.
3. Results and discussion
3.1. Representative HPLC chromatograms
In this study, a gradient elution method has been developed for
the separation of LJGTEs. In such systems, chromatograms of
reference standards and the typical sample of LJGTEs were shown
in Fig. 1. A good separation was achieved within 40 min using the
chromatographic conditions described. The most intense band was
the one for EGCG. In addition, no other chromatographic peaks
were observed around the 180 min elution time (0–50 min using
the gradient conditions above, 50–180 min using 100% A) at the
given wavelength. A detection wavelength of 280 nm was selected
due to its stability compared to detection at 210 nm. The baseline
obtained at 210 nm was very unstable and difficult to balance, thus
it was difficult to achieve baseline separation (see S6 in Supporting
Information).
3.2. Validation of HPLC method
Validation of the method was required to assure the suitability
for its intended use. Different characteristics such as calibration
Fig. 1. Representative HPLC chromatograms of reference standards (A) and green
tea extract (B). Peak identification: 1. GA. 2. GC. 3. EGC. 4. CAF. 5. C. 6. EC. 7. EGCG. 8.
GCG. 9. ECG. 10. CG. It was obtained using the Thermo ODS hypersil column and
Wufeng HPLC instruments, the similar chromatogram could be obtained using
other columns and instruments.
1115
curve, precision, accuracy and specificity were investigated accord-
ing to the USP32 <1225 VALIDATION OF COMPENDIAL PROCEDURES>
(United States Pharmacopeia, 2009).
The evaluation results of the calibration model were shown in
S7 (in Supporting Information). Using Student’s t-test, the intercept
and the regression coefficient were investigated. Both the calibra-
tion line and the residuals were graphically inspected and evalu-
ated. The correlation coefficient for all trials was at least 0.999.
The graphical inspection showed a linear correlation and the high-
est residual of 4% was still lower than the limit of 5%. It could be
concluded that a linear model was applicable in the concentration
range as described in S7 (in Supporting Information).
The precision experiment mainly included the investigation of
the repeatability and the precision. Six independent samples on
three different days were analyzed by the same analyst. The results
in S7 obtained on different days were not statistically different.
Since the RSD values for intra-day precision and inter-day precision
were of the same order of magnitude and less than 3%, the method
could be considered precise.
The accuracy was investigated by performing a recovery exper-
iment. The results of the recovery experiment were shown in S7
(see Supporting Information). The average of the recovery for all
ten components was (97.6–101.5)% with an RSD of below 1.9%. It
was shown that the recovery was not statistically different from
100%, which indicated that the method was accurate.
The specificity of the HPLC method was investigated by check-
ing mean retention time and the peak purity of green tea extracts.
This was performed by comparing the UV spectrum of the peak in
the reference standard and in the green tea extract. No deviations
were observed during the comparison of the UV spectra of the
peaks in the reference standard and in green tea extracts. The SD
of mean retention time was all less than 3%. The results indicated
that no interference occurred by other constituents in the solution.
The limit of detection (LOD) was determined at a signal-to-
noise ratio of 3:1. The limit of quantification (LOQ) was determined
at a signal-to-noise ratio of 10:1. The data of LOD and LOQ were
listed in S7.
3.3. Robustness test
To apply the determination method in different laboratories,
the analysts, instruments and columns were inevitable to change.
The different types of columns with different carbon load, surface
area and end capping may affect the results of the tailing factor
or symmetry factor, resolution and theoretical plate number. The
different HPLC instruments and different detectors affected the cal-
culation of peak area which mainly related with slit of detector, the
bandwidth value, the slope sensitivity and peak width and ways of
integration. The accidental errors caused by different analysts
acted sometimes in one and sometimes in another direction and
by their very nature can’t be predicted. Thus the potential influ-
ences should be fully considered in order to better control the
related parameters.
Taguchi Design: Taguchi Design also called Robust Design, is a
scientific and effective approach for the robust design of multi-
factor experiment (Peng, 2013; Wang, Sun, & Liu, 2014). In this
test, to evaluate the fluctuations and stability of QAMS, Taguchi
Design – Static design (Nominal is the best) was performed
(Table 2). Given the range of available reversed-phase stationary
phases and HPLC instruments, the representative columns (S5)
and HPLC instruments systems (S4) were selected. Signal-to-
noise (S/N) were introduced as evaluation parameters to consider
the influence of the mean and variance together. The target value
was the true fx of other nine components, the signal factor was
the measured fx of other nine components, six columns, three LC
instruments and three analysts were set as the controlling factors,
1116 D.-W. Li et al. / Food Chemistry 197 (2016) 1112–1120

and the noise was the fluctuations of the results changing with the
circumstances which were difficult to control. Whether the results
were around the target value was the major consideration. From
the results of residual plots in Fig. 2, it can be seen that all the
numerical points were presented as linear distribution (Fig. 2A),
which means that the results could be judged as normal distribu-
tions. And also its characteristic in Fig. 2B was similar to ‘‘bell”
shaped curve, which means that data collections were reasonable
and adequate and the process design was stable. The results were
reliable. From Fig. 2C and D, it can be seen that all the results were
distributed randomly, which showed that there were no abnormal
values in the text.
The results in S8 (in Supporting Information) clearly showed
that the factor of column (P < 0.05) had more important influence
to the mean value. The factor of column and the instrument had
more important influence (P < 0.05) to S/N. The results were consis-
tent with the response results in S9 and the main effects plot in
S10. For mean value, the column was the most significant factor
(Delta = 0.084), the analyst was intermediate (Delta = 0.029), the
instrument was least affected (Delta = 0.026). For S/N, the column
was also the most significant factor (Delta = 0.421), the instrument
was intermediate (Delta = 0.177), the analyst was least affected
(Delta = 0.096).
Intuitive analysis: The results in Table 2 showed that the mean
fx for all the nine components on all columns and instruments by
different analysts were 0.54, 6.79, 7.37, 0.55, 1.81, 1.87, 0.88,
0.69, and 0.69 respectively. And the RSDs for all were 1.75%,
3.18%, 4.47%, 2.44%, 2.93%, 2.86%, 3.67%, 1.15%, and 1.12% respec-
tively. It can be seen that the deviations of fx values for all the nine
components were 65%. The influences of stationary phase, LC
instrument and analyst on the results of fx for nine compounds
were further studied by Intuitive analysis. Through Intuitive anal-
ysis, the main factors that affect the fx may be found. The analysis
of range (R) in S11 (in Supporting Information) showed the impor-
tance of factors effected on fx of GC were column > instru-
ment > analyst. While on fx of EGC, the sequence was
column > analyst > instrument. The effect of above elements on
the other 7 components was small. Altogether, the range results
showed that the errors caused by different instrument, analyst
and column were small. This provides the foundation for further
examining the stability of fx.
Variance analysis: Based on the Intuitive analysis results, the
significance level of variance analysis for the column, instrument
and analyst were given. For the stationary phase factors, the results
in S12 (in Supporting Information) clearly showed that the factor
of column had more important influence than the factor of analyst
and instrument and the column was the most significant factor
(P < 0.05) especially for the component GA, GCG, ECG and CG.
The column had the most significant influence on the results and
should be well controlled.
Correlation analysis: In order to describe the possible relation-
ship of the fx results of nine components, the correlation analysis
was made based on the results in Table 2. From the results in
S13 (in Supporting Information), the Pearson Correlation between
C and EC was 0.870, the Pearson Correlation between ECG and CG
was 0.851. This indicated that there is a strong correlation of fx
between C and EC, ECG and CG. The results were very consistent
with the very similar compound structure of C and EC, ECG and
CG. The Pearson Correlation between GC and EGC was 0.152, but
the Sig. (2-tailed) was 0.546, which indicated that GC and EGC
had some similar relevance. What’s more, the Pearson Correlation
or the Sig. (2-tailed) was large among different compounds such
as GA and C (0.675), GC and EC (0.624), EC and ECG (0.771). The
reason may be that the molecular structure of these compounds
had some correlations during HPLC detection because of the influ-
encing factors such as solvent effect, conjugate effect, chro-
mophore (auxochrome), absorption band or electronic transition.
Such factors act together to produce the similar fx.
Based on the above analysis results, it can be seen that the con-
version factors were more prone to be affected by the column. The
column had the most significant influence on the results. Still, the
errors caused by different instrument, analyst and column were
small. Maybe it was because different instruments such as sensitiv-
ity, integration or other parameters and different columns, analysts
were different, but if the different compounds were detected in the
same instrument and column, the influence might be more or less
identical, the results of fx would not differ a lot. Although fx was
inevitable to change under different analysts, instruments, or col-
umns, these changes did not affect the fx results significantly and
the range of its fluctuations could be acceptable. QAMS could be
applied in different laboratories with carefully controlling the
parameter of column.

Table 2
The results of fx for nine compounds by 18-run Taguchi Design – Static design⁄.

Run order Stationary phase LC instrument Analyst EGCGa GA GC EGC CAF C EC GCG ECG CG
1 GRACE Agilent 1 1 0.53 6.52 7.51 0.56 1.74 1.87 0.86 0.68 0.67
2 GRACE Wufeng 2 1 0.53 6.56 7.40 0.56 1.75 1.85 0.87 0.68 0.67
3 GRACE Shimadzu 3 1 0.53 6.68 6.92 0.54 1.81 1.87 0.89 0.69 0.68
4 APROMA Agilent 1 1 0.55 6.83 7.61 0.57 1.81 1.86 0.88 0.69 0.68
5 APROMA Wufeng 2 1 0.56 7.02 7.79 0.57 1.82 1.86 0.88 0.69 0.68
6 APROMA Shimadzu 3 1 0.55 6.77 7.23 0.53 1.79 1.82 0.86 0.70 0.69
7 Hypersil Agilent 2 1 0.55 6.98 7.45 0.56 1.83 1.87 0.89 0.71 0.70
8 Hypersil Wufeng 3 1 0.54 6.79 7.29 0.57 1.85 1.88 0.90 0.71 0.70
9 Hypersil Shimadzu 1 1 0.55 6.47 7.59 0.57 1.92 1.96 0.91 0.69 0.69
10 Agilent Agilent 3 1 0.53 6.96 6.88 0.54 1.79 1.86 0.89 0.69 0.69
11 Agilent Wufeng 1 1 0.54 6.72 6.74 0.55 1.85 1.89 0.93 0.69 0.68
12 Agilent Shimadzu 2 1 0.56 6.31 7.21 0.56 1.90 1.99 0.93 0.69 0.69
13 Phenomenex Agilent 2 1 0.53 7.08 7.81 0.54 1.70 1.76 0.84 0.69 0.69
14 Phenomenex Wufeng 3 1 0.53 6.89 7.94 0.54 1.75 1.78 0.84 0.69 0.69
15 Phenomenex Shimadzu 1 1 0.54 7.00 7.12 0.55 1.83 1.91 0.92 0.69 0.68
16 Kromasil Agilent 3 1 0.54 7.03 7.21 0.54 1.80 1.86 0.84 0.70 0.69
17 Kromasil Wufeng 1 1 0.54 6.84 7.43 0.54 1.79 1.86 0.84 0.70 0.69
18 Kromasil Shimadzu 2 1 0.54 6.74 7.54 0.54 1.79 1.87 0.84 0.70 0.69
Means 1 0.54 6.79 7.37 0.55 1.81 1.87 0.88 0.69 0.69
SD 0 0.01 0.22 0.33 0.01 0.05 0.05 0.03 0.01 0.01
RSD (%) 0 1.75 3.18 4.47 2.44 2.93 2.86 3.67 1.15 1.12
⁄
Obtained by Minitab 16, DOE, Taguchi Design, L18 (61
32).
a
EGCG was used as a quality indicator.
 D.-W. Li et al. / Food Chemistry 197 (2016) 1112–1120
Fig. 2. Residual plots for mean value.
3.4. The system suitability and chromatographic peaks identification
The system suitability was evaluated via tailing factor, theoret-
ical plate number and resolution (S14 in Supporting Information).
The theoretical plate number ranged from 2667 to 94,488, respec-
tively. The resolutions of the ten compounds were >1.5, which
demonstrated that the compound peaks were not disturbed by
adjacent peaks in the quantization of their amounts. The tailing
factor of the compounds peaks ranged from 0.74 to 1.3, indicating
the absence of severe peak fronting or tailing.
For better authentication as well as convenience to quality con-
trol of the commercial LJGTEs, the relative retention time (RRT) of
the other nine components (relative to EGCG) by different
columns, instruments and analysts (Table 2) was used to identify
its chromatographic peak position. EGCG was explicitly identified
and designated as the reference peak in the LJGTEs samples. The
mean value of RRT relative to EGCG was 0.20 ± 0.02 (GA),
0.26 ± 0.03 (GC), 0.42 ± 0.03 (EGC), 0.53 ± 0.02 (CAF), 0.60 ± 0.03
(C), 0.91 ± 0.03 (EC), 1.24 ± 0.05 (GCG), 1.63 ± 0.05 (ECG),
1.74 ± 0.05 (CG), respectively (mean ± SD). The results showed that
RRT was stable and could be used to identify the other nine com-
pounds referring to EGCG in despite of the differences in columns
or HPLC instruments, or other factors in different laboratories.
Besides, the chromatographic peak area relative to EGCG or ECG
which was obviously different in the chromatograms of LJGTEs,
could also be an efficient way for identification and location.
3.5. The assessment of QAMS method
In order to evaluate and verify QAMS feasibility for the determi-
nation of multi-compounds in LJGTEs, the contents of eight tea cat-
echins, GA and CAF were determined by ESM and QAMS in 26
batches, respectively. The linear regression model was built
between the two variables, to examine the deviations between
QAMS and ESM. The independent variable was determined by
QAMS and the dependent variable was determined by ESM. All
the statistically significant coefficients in the regression model
1117
(S15 in Supporting Information) were 0.000, and the model yielded
an R2 value of 0.994, 0.990, 0.983, 0.991, 0.997, 0.987, 0.996, 0.976
and 0.986, respectively. The results implied that there was good
statistically significant correlation between the two variables,
and there were no significant differences in the content results
obtained by QAMS and ESM.
The results could be predictable from Eqs. (1) and (2). This is
because under the reliable chromatographic conditions, the nature
of the calculation of fx was the intrinsic function and proportion
relationship between the selected reference analyte and other ana-
lytes. The various errors related with instruments, columns, ana-
lysts, reagents, experimental methods or environment conditions
had been considered in the process of calculation of fx. Thus if
the deviations of fx in different columns, instruments and analysts
were small, the differences between QAMS and ESM would be
small as well. On the other hand, large variations in fx would result
in large differences in the content results. The deviations also came
from the differences between the average fx and the single fx
obtained from the Thermo ODS hypersil column and Wufeng
instrument. Although using the same fx may obtain a smaller dif-
ference, to check the differences in different laboratory between
QAMS and ESM, the average fx was used for the calculation of the
content of nine components. With the comparisons of the content
of all the nine components in LJGTEs by ESM and QAMS (see S16 in
Supporting Information), it was shown that all the contents were
located within the vicinity of linearity y = x (Fitting degree:
R2 = 0.9991). The data showed that using EGCG as reference for cal-
culation by QAMS, the contents variations were much lower than
the quantification error. Their contents variations within the range
of 5% which was the requirement of Pharmacopoeia indicated that
QAMS can be applied in the determination of eight tea catechins,
GA and CAF in different labs.
3.6. Quality evaluation of LJGTEs by QAMS
The contents of eight tea catechins, GA and CAF in 26 batches of
LJGTEs obtained by QAMS were listed in Table 1. It was observed
1118 D.-W. Li et al. / Food Chemistry 197 (2016) 1112–1120
Fig. 3. Dendrograms of hierarchical cluster analysis for the 26 tested samples of LJGTEs. The abscissa indicates the Euclidean distances, whereas the ordinate expresses the
sample numbers.
that among the ten components, the mean content of GA
(16.2 mg/g), CAF (10.9 mg/g), C (10.5 mg/g) and CG (5.0 mg/g)
accounted for lower content in LJGTEs. Conversely, the mean con-
tent of EGC (183.9 mg/g), EGCG (445.9 mg/g) and ECG (102.8 mg/g)
was in higher content. For all the 26 batches, the analyte of highest
Fig. 4. Icicles for the 26 tested samples of LJGTEs.
concentration was EGCG (524.6 mg/g) in producers No. s9 and the
analyte of lowest concentration was CG (1.2 mg/g) in No. s21 and
No. s22 and No. s23. The maximum total content of 10 compounds
was 982.2 mg/g, while the minimum total content of 10
compounds was 730.1 mg/g.
The data in Table 1 as discussed above revealed differences
among various sample pairs. To show the clear classification of
the samples and the relationships among the samples and evaluate
the variation of LJGTEs, HCA and CDA were performed using the
IBM SPSS Statistics 19.0 software (IBM, USA) (Alaerts et al., 2014;
An, Wang, Lan, Hashi, & Chen, 2013; He, Zhu, Goda, Cai, &
Komatsu, 2014; He et al., 2009; Kim, Shin, & Seo, 2014; Kong
et al., 2014). HCA can efficiently produce a qualitative and quanti-
tative representation of the original experimental results through
statistical organization and graphical display distinguishing differ-
ent herbal species in TCMs. The dendrograms (Fig. 3) and icicles
(Fig. 4) were constructed according to HCA based on the squared
Euclidean distance. It showed the relationships and distribution
patterns among the LJGTEs samples collected from seven produc-
ers of different batches from different origins. The contents of eight
principal bioactive compounds in 26 batches of commercial LJGTEs
samples were selected as the clustering variable.
The dendrograms clearly showed that the 26 tested samples of
LJGTEs were divided into three main clusters. Cluster one consisted
of nine samples collected from producers LWP, SOP. The icicles
clearly showed that these samples could be further divided into
 D.-W. Li et al. / Food Chemistry 197 (2016) 1112–1120
three groups (S17; S21; the rest, respectively). Cluster two consisted
of 14 samples collected from producers ZGP, XGP, STP, HSP, and SSP.
Cluster three consisted of three samples collected from producers
SSP. The cluster results were consistent with the icicles. The classi-
fication of results coincided well with the geographical origin of the
samples. Longjing green tea was mainly divided into two groups
based on origin: West-lake Longjing and Zhejiang Longjing. West-
lake Longjing was distributed in the West Lake District in Hangzhou,
while Zhejiang Longjing was mainly distributed in Qiantang and
Shaoxing District. Group one belonged to West-lake Longjing. Group
two belonged to Shaoxing Longjing. Lastly, group three belonged to
Qiantang Longjing. The samples produced by SSP were from two dif-
ferent origins: Qiantang and Shaoxing District. The results indicated
variations in component compositions and quantities were the
result of different origins. The main difference between the different
clusters was the source of Longjing green tea. A possible explanation
was that the different origins of Longjing green tea had been culti-
vated in different environments or in a different manner. These fac-
tors include soil type, climate, harvesting time, harvesting process,
storage condition and cultural practices. The harvesting time of
Longjing green tea was mainly concentrated in spring and summer.
The differences and similarity between different samples were also
shown in approximate matrix table (squared Euclidean distance)
(see S17 in Supporting Information). The values in the table repre-
sented the similarity coefficient between each sample, in which
the larger value represented a greater difference and the similarity
was relatively low.
Furthermore, CDA was introduced to create variables by taking a
special linear combination of the original variables. Based on the
results of HCA, Longjing green tea was divided into three categories
based on origin: West-lake Longjing, Qiantang Longjing and Shaox-
ing Longjing. The canonical variables of eight catechins were cre-
ated so that they contained the most useful information in a set
of original variables. To a certain extent, CDA may illustrate the dis-
tances between the classifications under investigation in a reduced
dimensional space. This is shown in S18 of the Supporting Informa-
tion where the three categories were significantly different from
each other. For new samples of LJGTEs, with the results obtained
by QAMS and HCA, it only needs to input the corresponding posi-
tion in the joint distribution map; the map would show the new
location of data. Through the position, which was a classification,
it can be seen which classification it belonged to and the differences
between each. The presented method suggested that the quality of
different LJGTEs samples were not consistent, and it may be helpful
to group and classify different Longjing green tea according to their
cultivation and origin. It was observed that the QAMS strategy was
accurate, stable, feasible and credible. Based on the reliable chro-
matographic conditions, by the determination of EGCG which was
easy to be obtained, QAMS results can be applied in the content cal-
culation of other nine components and in the quality control and
evaluation of LJGTEs in different laboratories.
Acknowledgments
The authors would like to thank Skyherb Ingredients for the col-
lections of different batches of Longjing green tea extracts for this
study. The authors are grateful to analysis and measurement of Zhe-
jiang University for providing MS and NMR data. This project is
financially supported by the Chinese Pharmacopoeia Commission.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.
11.101.
1119
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