Food Control xxx (xxxx) xxx

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Inductively coupled plasma mass spectrometry (ICP-MS) and inductively

coupled plasma optical emission spectrometer (ICP-OES)-based
discrimination for the authentication of tea
Hong-lin Liu a, b, Qing Meng a, Xin Zhao b, Yu-long Ye c, Hua-rong Tong a, *,
a
College of Food Science, Southwest University, Chongqing, 400715, China
b
Chongqing University of Education, Chongqing Collaborative Innovation Center for Functional Food, Chongqing Engineering Research Center of Functional Food,
Chongqing Engineering Laboratory for Research and Development of Functional Food, Chongqing, 400067, China
c
Tea Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, 610066, China

A R T I C L E I N F O A B S T R A C T

Keywords: Multielement and stable isotopes analysis combined with chemometrics as a potential tool for first authenticity
Tea discrimination of teas. Eighty-four statistically significant elements and stable isotopes from Chongqing tuo tea
Discrimination with 4 formulations were determined using ICP-MS and ICP-OES. Four chemometrics were used to build
Chemometrics
discrimination models.The discrimination of PLS-DA and LDA were better than that of HCA and PCA. 114Cd,
Formula 95
ICP-MS
Mo, 85Rb, Co, 133Cs and P were the most important markers for successful authenticity discrimination of teas.
ICP-OES Under the validation by cross-validation, test sets and blind data sets, the precisions of prediction of the PLS-DA
and LDA were not less than 90%. The LDA performs best, with precisions of prediction of 100%. This study helps
to promote the application of fingerprint technology of multi-elements and stable isotopes in combination with
chemometric analysis as a very promising tool for a worldwide inspection of the authenticity of teas.

1. Introduction authenticity of teas to protect the interests of consumers and producers.

Food safety has attracted worldwide attention, and one of its
important aspects is food authenticity. In general, food adulteration,
mislabeling, and origin mismatch are the main parameters that affect
food authenticity (Chen et al., 2014). Tea is one of the most common
drinks in the world, which has beneficial health effects due to its anti­
oxidant properties (Liu et al., 2019; Liu, Xing, & Fei, 2008). Tea blended
is a commonly used method to improve and stabilize its quality, expand
commodity supply, increase quantity, obtain higher economic benefit.
Therefore, tea prices should vary from recipe to recipe. Sensory quality
has not a sharp distinction among similar products, so unscrupulous
merchants may fake the labels of their products. This condition has not
only eroded consumer confidence but also significantly damaged the
reputation of international trade (Zhao, Zhang, & Zhang, 2017).
Chongqing tuo tea is blended from high-quality teas from several
tea-growing regions in Chongqing and Yunnan, China. The sensory
qualities among Chongqing tuo tea are similar, so consumers cannot
identify the quality of Chongqing tuo tea. Therefore, It is necessary to
develop a reliable and accurate method for analyzing and verifying the
Near infrared spectroscopy (NIR) (Firmani, De Luca, Bucci, Marini, &
Biancolillo, 2019; Zhou et al., 2009), high-performance liquid chroma­
tography (HPLC) (He et al., 2015; Wang et al., 2014), gas chromatog­
raphy mass spectrometry (GC-MS) (Ye, Zhang, & Gu, 2012), and
colorimetric artificial tongue and nose methods (Huo, Wu, & Yang,
2014) have been used to determine tea components, such as caffeine and
catechins. The contents of these compounds vary with the conditions of
origin, processing, and storage because of changes in oxygen density,
temp, and humidness (Han, Bae, & Hyon, 2009; Teshome, Debela, &
Garedew, 2013).The contents of multielement and stable isotopes can
also reflect differences in plant growth conditions. The compositions of
elements and stable isotopes are lower affected by machining and
stockpile, and the content is more stable. Multielement and stable iso­
topes are formed by the comprehensive action of geographical sources,
varieties, and climate (e.g., precipitation, temperature, and sunshine
time) (Dai et al., 2015; Wang et al., 2011). These theory provides a
possibility for authenticity discrimination of tea with multielement and
stable isotopes.
Chemometrics are common methods for discrimination of food.

* Corresponding author.
E-mail address: huart@swu.edu.cn (H.-r. Tong).

https://doi.org/10.1016/j.foodcont.2020.107735
Received 12 July 2020; Received in revised form 28 September 2020; Accepted 27 October 2020
Available online 30 October 2020
0956-7135/© 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Hong-lin Liu, Food Control, https://doi.org/10.1016/j.foodcont.2020.107735
H.-l. Liu et al.
Hierarchical clustering analysis (HCA) is an unsupervised chemometric
which demonstrates the correlation between variables and samples
(Yudthavorasit, Wongravee, & Leepipatpiboon, 2014). Principal
component analysis (PCA) is also an unsupervised pattern recognition
chemometric that reduces dimension of data and provides a local view
and trend of spatial data (Stanimirova, Daszykowski, & Walczak, 2007).
Partial least squares discriminant analysis (PLS-DA) is a regression
discriminator which predict a group of variables from a large number of
independent variables and identify them as function classes (Wold,
Ruhe, Wold, & Dunn, 1984; Chung et al., 2019). Linear discriminant
analysis (LDA) tests the affinity of each sample to the previously defined
group by minimizing intra-group variance and maximizing inter-group
variance (Moncayo, Manzoor, & Caceres, 2015). The use of multiele­
ment and stable isotopes in combination with chemometrics, such as
HCA, PCA, PLS-DA and LDA can feasibly and accurately identify the
geographic origins of teas (Koch et al., 2018; Li et al., 2018; Ma et al.,
2016; Pilgrim, Watling, & Grice, 2010; Rajapaksha et al., 2017; Zhao
et al., 2017).Unfortunately, reports on the authenticity discrimination of
tea are limited. And multi-element stable isotopes in tea have not been
reported.
We emphasized the surface features of 88 elements and stable iso­
topes of Chongqing tuo tea using inductively coupled plasma mass
spectrometry (ICP-MS) and inductively coupled plasma optical emission
spectrometer (ICP-OES).We discriminated authenticity of tea among
four different formulas with multielement and stable isotopes in com­
bination with 4 chemometrics analysis. We compared the discrimination
performance of the chemometric approaches, and selected markers in
order to successful authenticity discrimination of teas.We expect that
the preliminary results will demonstrate the feasibility of using our
authentication technology to discriminate authenticity of tea in China.
The ultimate goal is to prevent the false labeling of tea and possibly
apply the proposed method to other tea worldwide.We want to screen
out the majorization chemometric approach and chemical markers of
authenticity discrimination of other foods.
2. Materials and methods
2.1. Tea samples collection
In 2019, we collected 4 batches of Chongqing tuotea with diverse
formulas from the same factory (n = 27 for each batch). And the tea
samples selected for the experiment successively named as Chongqing
tuotea 1, 2, 3 and 4. Because of the inhomogeneous mixture in course of
manufacture of tea samples and different growth points of tea raw ma­
terials, we stochasticly extracted 27 samples from each batch of
Chongqing tuo tea. Matching plans of Chongqing tuo tea 1, 2, 3, and 4 of
tea blend raw materials were ascribed in detail (Table S1). Their
facticity, traceability, and the same production system of these products
were ensured by the country produce quality control (QC) service
agency in China before they sell at retail market. An additional
Chongqing tuo tea 1, 2, 3, and 4 samples (n = 12 for each batch) were
also collected for model verification (not used for model practicing),
only one serial number were tagged for blind sample processing.
2.2. Sample preparation
All tea samples are unpacked and placed in a dry tin at room tem­
perature for a week to ensure the same water content (~5%). The tea
was grounded by ball crusher (MM301, Retsch Corporation, Germany),
and passed through 100 mesh sieve. 0.2 g of every ground tea specimen
were exactly weighed (exact to 0.0001g), and put in a polytetrafluoro­
ethylene (PTEF) digestion container. The PTEF digestion container was
steeped with 20% nitric acid (HNO3) overnight, which was washed with
ultra-pure water until no tart flavour taste was presented and dried
thoroughly before usage. Afterwards, the samples were treated with a
mixture 5 mL of nitric acid (65% w/w, Merck company) and 2 mL of
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Food Control xxx (xxxx) xxx
hydrogen peroxide (30% w/w, Merck company). Thermal digestion of
the samples were carried out in a microwave digestion device (MARS 6
CEM, USA, Matthews) according to the set microwave digestion pro­
gram (Table 1). The digested samples were cooled to room temperature
and taken out, and slowly opened the digestion tank cover under the
fume hood to exhaust the gas. Then the digestion tanks were put on an
acid extraction heater (bhw, China, Botong) at 140 ◦ C for acid extraction
for 2 h. Then we taked out the digestion tank, washed the digestion tank
and covered with a small amount of ultra pure water 3–4 times into the
PTEF volumetric flask until they are cleaned. And finally, we diluted the
washing solution to a 50 ml PTEF volumetric flask for standby.
2.3. Multielement and stable isotope analysis by ICP-MS and ICP-OES
The concentrations of multielement and stable isotopes were deter­
mined of the standby liquids by ICP-MS (PerkinElmer NexION 300X,
USA) and ICP-OES. The required requirements and survey parameters of
the instrument were shown in Table S2. The internal standards (Re, In,
Rh) and tuned liquors (Be, Ce, Fe, In, Li, Mg, Pb, and U) were corrected
matrix effects and compensated for possible changes in apparatus
function during measurement. The concentrations of multielement and
stable isotope were determined in kinetic-energy-discrimination (KED)
mode in ICP-MS. ICP-OES were made using a synchronous ICP spec­
trometer (Agilent 5100, USA) equipped with a solid state detector.
Samples were atomized by an ultrasonic atomizer with a solventia sys­
tem (U-5000 AT, CETAC Technologies, Omaha, NE).
2.4. Statistical analysis
Eighty-eight elements and stable isotopes were analyzed for each
sample. Three replicates survey were performed if the mean standard
deviation of the replicates beyond expected survey error (≈±1‰).
Multiple comparisons used Duncan post test and the differences were
possessed statistically significant at the standard of p < 0.05, for 95%
confidence interval. SPSS statistical software (Version 23.0, IBM, USA)
was used for one way ANOVA and LDA.SIMCA software (version 13.0,
Umetrics, Umeå, Sweden) was used to apply HCA, PCA and PLS-DA. All
variables are automatically labeled (autoscaled) before the HCA, PCA,
PLS-DA, and LDA are applied (Borges, Gelinski, Souza, Barbosa, &
Batista, 2015).
3. Results and discussion
3.1. Analytical performance
The linearity, sensitivity, accuracy, and precision of the ICP-MS and
ICP-OES methods were assessed in order to analyze Chongqing tuo tea
according to the EURACHEM guidelines (Eurachem, 2014) (Table S3).
The good linearity of analysis curve was r2 > 0.999. The sensitivity was
determined based on the detection (LOD) and quantitative limits (LOQ)
as below: LOD = 3Sy/S, LOQ = 10Sy/S, where Sy is the standard de­
viation (SD) of the respond, and S is the rake rat of the analytical curve.
In the Chongqing tuo tea, the LOQ values of various multielement and
stable isotopes were far lower than their natural levels, which ensured
the reliability of their concentration determination. The CRM of tea
(GBW10016, GSB-7) was analyzed (n = 3) based on the accuracy of the
ICP-MS and ICP-OES methods. Acceptable mean recoveries within
Table 1
Program of microwave digestion.
Steps Rise Time (min) Hold-up Time (min) Temperature (◦ C) Power
(W)
1 20 10 120 1400
2 10 10 160 1400
3 10 30 180 1400
H.-l. Liu et al. Food Control xxx (xxxx) xxx

92.6%–107.9% were observed for the chemical species, suggesting a
good accuracy (Codex Alimentarius, 2009). The precision of the two
methods was quantified based on the relative standard deviation (RSD
%) surveyed in the reanalysis of the samples. These values are less to
15% for multielement and stable isotopes, which can be considered an
acceptable limit for ICP-MS and ICP-OES analysis of tea (Altundag &
Tuzen, 2011; Dolan & Capar, 2002; D’Archivio et al., 2019). In
conclusion, the ICP-MS and ICP-OES were allowable concentration of
multielement and stable isotopes used in the authenticity discrimination
of Chongqing tuo tea.

Fig. 1. Pearson correlation analysis and hierarchical clustering analysis between variables and samples. The correlation coefficient is indicated by the intensity of the
colors, as indicated by the color scale. (1, 2, 3, 4 represent Chongqing tuo tea1, Chongqing tuo tea 2, Chongqing tuo tea 3, and Chongqing tuo tea 4, respectively).
(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3
H.-l. Liu et al.
3.2. Multielement and stable isotopes fingerprints of Chongqing tuo tea
Results of the means, SD and scope of the multielement and stable
isotopes composition analysis of Chongqing tuo tea are shown in
Table S4. The concentrations of multielement and stable isotopes in
Chongqing tuo tea were greater than 1 g kg − 1 for K, P, Ca, Mg, and Si.
The order of the mean multielement level is as follows: K > P > Ca > Mg
> Si. Hence, the predominant multielement and stable isotope in all
Chongqing tuo tea samples is K. And mean concentrations of Al, B, Cu,
Fe, Mn, Na, 137Ba, 138Ba, 79Br, 81Br, 52Cr, 53Cr, 58Ni, 60Ni, 85Rb, 86Sr,
88
Sr, 46Ti, 47Ti, 48Ti, 64Zn, 66Zn, and 68Zn were greater than 1 mg kg − 1.
The remaining elements and stable isotopes had mean concentrations
below 1 mg kg − 1. These results were consistent with previous studies
(Ashraf & Mian, 2008; Cao, Qiao, Zhang, & Chen, 2010; Chen, Yu, Xu,
Chen, & Shi, 2009; Karak & Bhagat, 2010; Liu, Zeng, Zhao, & Tong,
2020; McKenzie, Jurado, & De Pablos, 2010).
Based on the above analysis, the divergences of 88 elements and
stable isotopes in four batches of tea were studied. By analyzing, 84 of
the 88 multielement and stable isotopes had statistical significance (P <
0.05) for the tea with different formulas. Each batch of samples had
characteristic multielement and stable isotope concentration finger­
prints in Duncan multiple comparisons. For the Chongqing tuo tea 1,
137
Ba, 48Ti, and K concentrations were the highest. The concentrations
of Ba, 59Co, 74Ge, 98Mo, 93Nb, 58Ni, 60Ni, 118Sn, 120Sn, 51V, Cu, Fe, and
138
Si were supreme in Chongqing tuotea 2. Chongqing tuo tea 3 had su­
preme characterizations of 6Li, 7Li, 85Rb, 78Se, 80Se, 86Sr, 88Sr, 203Tl,
205
Tl, 89Y, 139La, 140Ce, 141Pr, 142Nd, 152Sm, 156Gd, 158Gd, 160Gd, 162Dy,
164
Dy, 165Ho, 169Tm, 172Yb, and 174Yb. The contents of 111Cd, 112Cd,
114
Cd, 52Cr, 53Cr, 95Mo, 64Zn, 66Zn, 68Zn, and P in Chongqing tuo tea 4
were the highest. However, significant differences were surveyed be­
tween the lowest and highest concentrations of each element or stable
isotope in same batch of tea (Table S4). Chemometrics should be
appraised the cluster trend of tea based on statistically significant
multielement and stable isotopes of the batch of different formulas for an
accurate authenticity discrimination of tea.
3.3. Exploratory statistical analysis
Eighty-four statistically significant elements and stable isotopes were
observed among four batches of tea (P < 0.05), which were adopted to
HCA and PCA methods for exploratory statistical analysis.
3.3.1. HCA
HCA described a five variable clusters (Fig. 1). Group 1 contained
121
Sb, 123Sb, 47Ti, As, K, Ca, and Mg. Group 2 contained Na, 6Li, 7Li,
151
Eu, 152Sm, 156Gd, 158Gd, 160Gd, Ce, 142Nd, Pr, La, Al, 203 Tl, 205Tl,
85
Rb, Cu, 96Mo, and 98Mo. Group 3 contained 200Hg, 202Hg, 46Ti, Sc,
71
Ga, V, 133 Cs, Mn, B, 206Pb, 207Pb, 208Pb, 86Sr, 88Sr, 154Sm, and 138Ba.
Group 4 contained 175Lu, 159Tb, 169Tm, 172Yb, 174Yb, Y, 162Dy, 164Dy,
165
Ho, 166Er, 168Er, 95Mo, 118Sn, 120Sn, Fe, 58Ni, 60Ni, Co, Si, 74Ge, Nb,
52
Cr, and 53Cr. The last group contained 180Hf, P, 70Ge, 72Ge, 48Ti, 137Ba,
69
Ga, 79Br, 81Br, 78Se, 80Se, 111Cd, 112Cd, 114Cd, 64Zn, 66Zn, 68Zn, 107Ag,
and 109Ag. Therefore, all rare-earth elements and stable isotopes can be
clustered. In general, the same family of elements in periodic table of
elements tend to be clustered, but elements in the same family can’t be
clustered all. The stable isotopes of most of the same element in the tea
samples can be clustered, but the stable isotopes of Ti, Sm, Mo, Ba, Ge,
and Ga can’t be clustered. The behavior of elements and stable isotopes
forming the cluster is similar, which is also made available for onions
(Ariyama, Nishida, Noda, Kadokura, & Yasui, 2006).
HCA also showed a sample clustering tree of 84 mineral elements and
stable isotopes concentration which described three samples clusters
(Fig. 1). Chongqing tuo tea 2 and 3 individually can be clustered,
respectively. Chongqing tuo tea 1 and 4 were mixed clustered.
Chongqing tuo tea with different formulas could not be accurate
authenticity discriminated by choosing 84 elements and stable isotope
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Food Control xxx (xxxx) xxx
concentration in HCA. Therefore, other chemometrics were further
searched to discriminate authenticity of tea with different formulas.
3.3.2. PCA
The results showed that Chongqing tuo tea from 4 batches with
different formulas can be discriminated in PCA. The cumulative variance
contribution rate of first three principal components (PCs) showed
28.8%, 15.5%, and 11.5% of overall variance (Fig. 2). However, they
were low (55.8%); thus, it is not a strong category. Chongqing tuo tea 1
appeared a association of positive and negative fractions of PC1 and
negative fractions of PC2. Chongqing tuo tea 2 exhibited negative
fractions of PC1 and positive fractions of PC2, while Chongqing tuo tea
3, which exhibited positive fractions in PC1 and positive and negative
fractions in PC2. Chongqing tuo tea 4 appeared a association of positive
and negative fractions of PC1 and PC2 (Fig. 2A). PC1 was greatly
affected by 205Tl, Y, 85Rb, 174Yb, and 172Yb. The main variables in PC2
were Co, V, 64Zn, 68Zn, and 66Zn (Fig. 2B).
Through the unsupervised methods of HCA and PCA, the four
batches tea were naturally divided into groups in the basic data matrix,
showing a good trend of sample grouping. But they couldn’t supply
message on the quality of clustering and confidence in clustering. Su­
pervised chemometrics were further searched to discriminate authen­
ticity of tea.
3.4. Supervised chemometrics modeling for sample authentication
Exactly 84 statistically significant elements and stable isotopes
(Table S4) were subjected to PLS-DA and LDA approaches for futher
authenticity discrimination of tea with different formulas.
3.4.1. PLS-DA
It was a suitable approach of PLS-DA for discriminating authenticity
of tea with different formulas (Fig. 3). A small overlap observed between
Chongqing tuotea 3 and 4 both existed in PLS-DA and PCA, which owed
to their similar computational theories. Our PLS-DA model showed good
pattern function (R2X = 0.686, R2Y = 0.952, and Q2 = 0.939) (West­
erhuis et al., 2008; Worley & Powers, 2013; Eriksson, Johansson,
Kettaneh-Wold, & Wold, 2001). This pattern expounds 68.6% of the
change of X and 95.2% of the change of Y, and a prediction capability Q2
of 93.9%.
The PLS-DA score graph had a good clustering, showing that the two
highest ranked R2X accounts for 43.7% of total variance (Fig. 3A). The
first R2X accounted for 28.7% of this variation, while the second R2X
accounted for 15.0% of this variation. In the first R2X, the largest con­
tributors were Cu and 205Tl, Chongqing tuo tea 2 could be discriminated.
The second R2X was mainly contributed by 137Ba and Co, Chongqing tuo
tea 1 could be separated from the other tea (Fig. 3B). The VIP values
explain a variable’s devotion in the projection, with a value of >1
usually used to discriminate variables which are major to the pattern.
Fifteen principal variables in PLS-DA of 95Mo, Al, Co, 133Cs, 86Sr, 88Sr,
Mn, P, 111Cd, 112Cd, 114Cd, 85Rb, La, 118Sn and 120Sn, were surveyed as
markers for authenticity discrimination of tea with different formulas.
To better test the stability of the PLS-DA model, 70% of tea of each
batches randomly selected from the total samples for training sets
(70%), model cross-validation (70%, the same with training dataset)
and test sets (the remaining 30%) for external validation in PLS-DA. The
recognition ability was represented by the percentage of tea correctly
discriminated during the model training process, and the overall accu­
racy was 95.8%. Chongqing tuo tea 1, Chongqing tuo tea 2 and
Chongqing tuo tea 4 could be discriminated clearly with accuracies of
100%, whereas the accuracy for Chongqing tuo tea 3 was 85.7%. The
overall accuracies of prediction were 93.1% and 91.7% during the
model cross-validation and test set, respectively. The accuracies of
Chongqing tuo tea 3 (84.2% during the model cross-validation and
80.0% during the model test set) and Chongqing tuo tea 4 (88.2% during
the model cross-validation and 88.9% during the model test set) lower
H.-l. Liu et al. Food Control xxx (xxxx) xxx

Fig. 2. PCA plots showing PC1, and PC2 effects to authenticate four batches of Chongqing tuo tea with different formulas. The ellipse on the score plots represents
the 95% confidence region for Hotelling’s T2. Score plot of PC1 and PC2(A), loading plot of PC1 and PC2 from PCA (B). (1, 2, 3, and 4 refer to refers Chongqing tuo
tea 1, Chongqing tuo tea 2, Chongqing tuo tea 3, and Chongqing tuo tea 4, respectively).

than those in model training (Table S5).
3.4.2. LDA
We further used LDA to discriminate the authenticity of Chongqing
tuo tea. The samples were divided into training sets (70%), model cross-
validation (70%, the same with training dataset) and test sets (the
remaining 30%) for external validation. The model was also appraised
by recognitional capacity with the training dataset and predictive power
with cross validation of model and test set, respectively. Discriminant
analysis showed that Chongqing tuo tea were well differentiated from
each other and the authenticity discrimination of Chongqing tuo tea was
quite good (Fig. 4 & Table S6). The recognitional capacity of the totality
exactness discrimination rate was 100%, predictive power of rate was
100% by both the cross validation model and test set, which were a
desirable value specially suitable for this method (Table S6).
After selecting the positive step-by-step variables according to Wilk’s
lambda criterion, 85Rb, Co, 133Cs, P, 114Cd, 58Ni, 205Tl, 95Mo, 48Ti, 121Sb,
46
Ti, K, Na, Si, and 207Pb were decomposed into a simplified model. The

5
H.-l. Liu et al. Food Control xxx (xxxx) xxx

Fig. 3. Comparison of PLS-DA results derived from 84 mineral elements and stable isotopes of four batches of Chongqing tuo tea with different formulas authenticity.
Score plot (A), and loading plot from PLS-DA (B). The ellipse on the score plots represents the 95% confidence region for Hotelling’s T2. (1, 2, 3, and 4 represent
Chongqing tuo tea1, Chongqing tuo tea 2, Chongqing tuo tea 3 and Chongqing tuo tea 4, respectively).

results indicated that two statistically significant discriminant Fisher
functions were formed, namely, Wilks’ lambda <0.001 (X2 = 1535.101,
df = 84, p < 0.001) for the first Fisher function and Wilks’ lambda
<0.001 (X2 = 908.328, df = 54, p < 0.001) for the second Fisher
function. The significant value of Wilks’ lambda value indicates that the
discriminant function is the basis of differentiation of the investigated
population. Both the first two functions accounted for 94.0% of total
variance. The first discriminant Fisher function expounded for 81.6% of
total variance, 85Rb and 205Tl contribute to it, and Chongqing tuo tea 2
and 3 could be discriminanted. Whereas the second discriminant Fisher
function expounded for 12.4%, 114Cd and Co are the key components of
it, and Chongqing tuo tea 4 could be distinguished ((Fig. 4 & Table S7).
Compared with PLS-DA, the separation in LDA was better. This might
caused by different algorithms between LDA and PLS-DA (Moncayo
et al., 2015). Among the variables selected by PLS-DA and LDA, all two
models considered 114Cd, 95Mo, 85Rb, Co, 133Cs and P as the most
important markers for authenticity discrimination of tea. The discrimi­
nation of PLS-DA and LDA were better than that of HCA and PCA.
3.4.3. PLS-DA and LDA models performance in blind datasets
To further examine the stabilization of the chemometrics applied for
calculation, a blind dataset of the Chongqing tuo tea blind samples (n =
12 for each batch) was classified using the constructed model. In
validation by the “blind” datasets, the totality accuracy rating of PLS-DA
and LDA were 91.7% and 100%, respectively (Table S8). The better
function of LDA indicated that the LDA approach is the most exact model
for authenticity discrimination of tea. Forty-four of 48 tea samples could
be predicted into the four groups correctly in PLS-DA (Table S8), except
that one Chongqing tuo tea 3 was wrongly classified into Chongqing tuo
tea 4 and three Chongqing tuotea 4 wrongly classified into Chongqing
tuotea 3.The good prediction performance (>90%) might be because of
different matching plans of Chongqing tuotea 1, 2, 3, and 4 (Table S1).
Therefore, the LDA models represented the best performance and
effectiveness of discriminating authenticity of tea with different
formulas.
3.5. Discussion
This study showed that the Chongqing tuo tea with each formula had
a typical characteristics of elements and stable isotope. Multielement
and stable isotope were also analyzed as a sign of hope to authenticate
the tea because their different blends of raw materials with different
environment.The multielement sections of soil varied in several envi­
ronmental and geographical factors (Zhao et al., 2017). Certain element
and stable isotope contents in the soil of Yunnan and Chongqing prov­
inces were different levels in China, leading to the accumulation of

6
H.-l. Liu et al.
Fig. 4. Authenticity discrimination of Chongqing tuo tea with different formulas based on 84 multi-elemental and stable isotopes by the LDA model.
multielement and stable isotopes in the Chongqing tuotea 1, 2, 3 and 4
(Wei, Zhang, Chen, & Wu, 1991). Transferring of multielement and
stable isotopes through the tea and soil has already been found and
quantified (Zhao et al., 2017). In addition, each formula of tea showed
significant differences in its multielement and stable isotopes due to the
different proportions of various tea raw materials. However, the transfer
of elements and stable isotopes in tea needs further study.
Using the technology of fingerprinting multielement and stable iso­
topes to identify the regions of tea has become a hot research topic. In
previous studies, Pilgrim et al. have verified the applicability of multi­
element for identification of tea from different regions and harvest times
(Koch et al., 2018; Li et al., 2018; Ma et al., 2016; Pilgrim et al., 2010;
Rajapaksha et al., 2017; Zhao et al., 2017). The results showed that on
the basis of multielement in combination with chemometric analysis, tea
samples could be discriminated. In our study,ICP-MS and ICP-OES
technology in combination with chemometrics were used for multiele­
ment and stable isotopes spectral analysis of tea, and authenticity of tea
could be reliably discriminated.
4. Conclusions
Quantitative fingerprinting of Chongqing tuo tea products contain­
ing 88 elements and stable isotopes were analyzed using ICP-MS and
ICP-OES. These technologies supply a fast and dependable tools for
producing enough abundant multielement and stable isotope finger­
printing. The results verified that 84 statistically significant elements
and stable isotopes could be used as fingerprints to authenticate the
Chongqing tuo tea with formulas. The discrimination results of the two
unsupervised approaches were worse than that of the two supervised
approaches in Chongqing tuo tea. On the basis of the raised PLS-DA and
LDA approaches, the authentication performance of LDA was the best.
114
Cd, 95Mo, 85Rb, Co, 133Cs and P were the most important markers for
successful discrimination of Chongqing tuo tea with their formulas. This
study announces the vast proficiency of multielement and stable iso­
topes for appraising of authenticity discrimination of food.
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CRediT authorship contribution statement
Hong-lin Liu: Conceptualization, Methodology, Software, Investi­
gation, Data curation, Writing - original draft, Writing - review & edit­
ing. Qing Meng: Software, Writing - review & editing. Xin Zhao:
Validation. Yu-long Ye: Resources, Methodology. Hua-rong Tong:
Supervision, Investigation, Project administration.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This study was supported by Southwest University and Chongqing
University of Education in 2019, Chongqing modern mountain charac­
teristics efficient agricultural tea industry technical system 2019-6,
2018-6; Project funded by Chongqing university innovation team con­
struction plan (CXTDX201601040).The authors thank the reviewers for
their perceptive and helpful comments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodcont.2020.107735.
References
Codex Alimentarius. (2009). Guidelines for the design and implementation of national
regulatory food safety assurance program associated with the use of veterinary drugs
in food producing animals. Codex Alimentarius. CAC/GL, 71, 22.
Altundag, H., & Tuzen, M. (2011). Comparison of dry, wet and microwave digestion
methods for the multi element determination in some dried fruit samples by ICP-
OES. Food and Chemical Toxicology, 49(11), 2800–2807. https://doi.org/10.1016/j.
fct.2011.07.064
H.-l. Liu et al.
Ariyama, K., Nishida, T., Noda, T., Kadokura, M., & Yasui, A. (2006). Effects of
fertilization, crop year, variety, and provenance factors on mineral concentrations in
onions. Journal of Agricultural and Food Chemistry, 54, 3341–3350. https://doi.org/
10.1021/jf0525481
Ashraf, W., & Mian, A. A. (2008). Levels of selected heavy metals in black tea varieties
consumed in Saudi Arabia. Bulletin of Environmental Contamination and Toxicology,
81, 101–104. https://doi.org/10.1007/s00128-008-9402-0
Borges, E. M., Gelinski, J. M. L. N., Souza, V. C. D., Barbosa, F., & Batista, B. L. (2015).
Monitoring the authenticity of organic rice via chemometric analysis of elemental
data. Food Research International, 77, 299–309. https://doi.org/10.1016/j.
foodres.2015.06.008
Cao, H. B., Qiao, L., Zhang, H., & Chen, J. J. (2010). Exposure and risk assessment for
aluminium and heavy metals in Puerh tea. The Science of the Total Environment, 408,
2777–2784. https://doi.org/10.1016/j.scitotenv.2010.03.019
Chen, H., Fan, C. L., Chang, Q. Y., Pang, G. F., Hu, X. Y., Lu, M. L., et al. (2014).
Chemometric determination of the botanical origin for Chinese honeys on the basis
of mineral elements determined by ICP-MS. Journal of Agricultural and Food
Chemistry, 62, 2443–2448. https://doi.org/10.1021/jf405045q
Chen, Y. X., Yu, M. G., Xu, J., Chen, X. C., & Shi, J. Y. (2009). Differentiation of eight tea
(Camellia sinensis) cultivars in China by elemental fingerprint of their leaves.
Journal of the Science of Food and Agriculture, 89, 2350–2355. https://doi.org/
10.1002/jsfa.3716
Chung, I. M., Kim, J. K., Yarnes, C. T., An, Y. J., Kwon, C., Kim, S. Y., et al. (2019). Fatty
acid- and amino acid-specific isotope analysis for accurate authentication and
traceability in organic milk. Journal of Agricultural and Food Chemistry, 67(2),
711–722. https://doi.org/10.1021/acs.jafc.8b05063
D’Archivio, A. A., Foschi, M., Aloia, R., Maggi, M. A., Rossi, L., & Ruggieri, F. (2019).
Geographical discrimination of red garlic (Allium sativum L.) produced in Italy by
means of multivariate statistical analysis of ICP-OES data. Food Chemistry, 275,
333–338. https://doi.org/10.1016/j.foodchem.2018.09.088
Dai, W. D., Qi, D. D., Yang, T., Lv, H. P., Guo, L., Zhang, Y., et al. (2015). Nontargeted
analysis using ultraperformance liquid chromatography-quadrupole time-of-flight
mass spectrometry uncovers the effects of harvest season on the metabolites and
taste quality of tea (camellia sinensis L.). Journal of Agricultural and Food Chemistry,
63, 9869–9878. https://doi.org/10.1021/acs.jafc.5b03967
Dolan, S. P., & Capar, S. G. (2002). Multi-element analysis of food by microwave
digestion and inductively coupled plasma-atomic emission spectrometry. Journal of
Food Composition and Analysis, 15(5), 593–615. https://doi.org/10.1006/
jfca.2002.1064
Eriksson, I., Johansson, E., Kettaneh-Wold, N., & Wold, S. (2001). Multi- and megavariate
data analysis principles and applications. Sweden: Umetrics AB: Umeå.
Eurachem. Eurachem guide. (2014). The fitness for purpose of analytical methods -A
laboratory guide to method validation and related topics. Eurachem, 978-91-87461-
59-0.
Firmani, P., De Luca, S., Bucci, R., Marini, F., & Biancolillo, A. (2019). Near infrared
(NIR) spectroscopy-based classification for the authentication of Darjeeling black
tea. Food Control, 100, 292–299. https://doi.org/10.1016/j.foodcont.2019.02.006
Han, D. K., Bae, J. Y., & Hyon, S. H. (2009). Biochemical and histological evaluations of
articular cartilages preserved in cold storage solution containing green tea catechin:
EGCG. Journal of Tissue Engineering and Regenerative Medicine, 6, 380–387.
He, X. Y., Li, J. K., Zhao, W., Liu, R., Zhang, L., & Kong, X. H. (2015). Chemical
fingerprint analysis for quality control and identification of Ziyang green tea by
HPLC. Food Chemistry, 171, 405–411. https://doi.org/10.1016/j.
foodchem.2014.09.026
Huo, D. Q., Wu, Y., & Yang, M. (2014). Discrimination of Chinese green tea according to
varieties and grade levels using artificial nose and tongue based on colorimetric
sensor arrays. Food Chemistry, 145, 639–645. https://doi.org/10.1016/j.
foodchem.2013.07.142
Karak, T., & Bhagat, R. M. (2010). Trace elements in tea leaves, made tea and tea
infusion: A review. Food Research International, 43, 2234–2252. https://doi.org/
10.1016/j.foodres.2010.08.010
Koch, W., Kukula-Koch, W., Komsta, L., Marzec, Z., Szwerc, W., & Glowniak, K. (2018).
Green tea quality evaluation based on its catechins and metals composition in
combination with chemometric analysis. Molecules, 23(7), 1689. https://doi.org/
10.3390/molecules23071689
Liu, J. P., Xing, J. M., & Fei, Y. T. (2008). Green tea (Camellia sinensis) and cancer
prevention:A systematic review of randomized trials and epidemiological studies.
Chinese Medicine, 3(1), 12. https://doi.org/10.1186/1749-8546-3-12
Liu, Z., Yuan, Y. W., Zhang, Y. Z., Shi, Y. Z., Hu, G. X., Zhu, J. H., et al. (2019).
Geographical traceability of Chinese green tea using stable isotope and multi-
Food Control xxx (xxxx) xxx
element chemometrics. Rapid Communications in Mass Spectrometry : Rapid
Communications in Mass Spectrometry, 33, 778–788. https://doi.org/10.1002/
rcm.8405
Liu, H. L., Zeng, Y. T., Zhao, X., & Tong, H. R. (2020). Improved geographical origin
discriminationfor tea using ICP-MS and ICP-OES techniques in combination with
chemometric approach. Journal of the Science of Food and Agriculture, 100,
3507–3516.
Li, L., Wen, B., Zhang, X. L., Zhao, Y., Duan, Y., Song, X. F., et al. (2018). Geographical
origin traceability of tea based on multi-element spatial distribution and the
relationship with soil in district scale. Food Control, 90, 18–28. https://doi.org/
10.1016/j.foodcont.2018.02.031
Ma, G. C., Zhang, Y. B., Zhang, J. Y., Wang, G. Q., Chen, L. Y., Zhang, M. L., et al. (2016).
Determining the geographical origin of Chinese green tea by linear discriminant
analysis of trace metals and rare earth elements: Taking Dongting Biluochun as an
example. Food Control, 59, 714–720. https://doi.org/10.1016/j.
foodcont.2015.06.037
McKenzie, J. S., Jurado, J. M., & De Pablos, F. (2010). Characterisation of tea leaves
according to their total mineral content by means of probabilistic neural networks.
Food Chemistry, 123(3), 859–864. https://doi.org/10.1016/j.foodchem.2010.05.007
Moncayo, S., Manzoor, S., & Caceres, J. O. (2015). Evaluation of supervised chemometric
methods for sample classification by laser induced breakdown spectroscopy.
Chemometrics and Intelligent Laboratory Systems, 146, 354–364. https://doi.org/
10.1016/j.chemolab.2015.06.004
Pilgrim, T. S., Watling, R. J., & Grice, K. (2010). Application of trace element and stable
isotope signatures to determine the provenance of tea (Camellia sinensis) samples.
Food Chemistry, 118, 921–926. https://doi.org/10.1016/j.foodchem.2008.08.077
Rajapaksha, D., Waduge, V., Padilla-Alvarez, R., Kalpage, M., Rathnayake, R. M. N. P.,
Migliori, A., et al. (2017). XRF to support food traceability studies:Classification of
Sri Lankan tea based on their region of origin. X-Ray Spectrometry, 46, 220–224.
https://doi.org/10.1002/xrs.2748
Stanimirova, I., Daszykowski, M., & Walczak, B. (2007). Dealing with missing values and
outliers in principal component analysis. Talanta, 72(1), 172–178. https://doi.org/
10.1016/j.talanta.2006.10.011
Teshome, K., Debela, A., & Garedew, W. (2013). Effect of drying temperature and
duration on biochemical composition and quality of black tea (Camellia sinensis L.)
O Kuntze at Wush, south western Ethiopia. Asian Journal of Plant Sciences, 12,
235–240. https://doi.org/10.3923/ajps.2013.235.240
Wang, L. Y., Wei, K., Cheng, H., He, W., Li, X. H., & Gong, W. Y. (2014). Geographical
tracing of Xihu Longjing tea using high performance liquid chromatography. Food
Chemistry, 146, 98–103. https://doi.org/10.1016/j.foodchem.2013.09.043
Wang, L. Y., Wei, K., Jiang, Y. W., Cheng, H., Zhou, J., He, W., et al. (2011). Seasonal
climate effects on flavanols and purine alkaloids of tea (Camellia sinensis L.).
European Food Research and Technology, 233, 1049–1055. https://doi.org/10.1007/
s00217-011-1588-4
Wei, F. S., Zhang, C., Chen, J. S., & Wu, Y. Y. (1991). Study on the background contents
on 61 elements of soil in China. Journal of Environmental Science-China, 12, 12–19.
Westerhuis, J. A., Hoefsloot, H. C. J., Smit, S., Vis, D. J., Smilde, A. K., VanVelzen, E. J. J.,
et al. (2008). Assessment of PLS-DA cross validation. Metabolomics, 4, 81–89.
Wold, S., Ruhe, A., Wold, H., & Dunn, W. J. (1984). Collinearity problem in linear
regression-The partial least squares (PLS) approach to generalized inverses. SIAM
Journal on Scientific and Statistical Computing, 5(3), 735–743. https://doi.org/
10.1137/0905052
Worley, B., & Powers, R. (2013). Multivariate analysis in metabolomics. Current
Metabolomics, 1, 92–107.
Ye, N. S., Zhang, L. Q., & Gu, X. X. (2012). Discrimination of green teas from different
geographical origins by using HS-SPME/GC-MS and pattern recognition methods.
Food Analytical Methods, 5(4), 856–860. https://doi.org/10.1007/s12161-011-9319-
9
Yudthavorasit, S., Wongravee, K., & Leepipatpiboon, N. (2014). Characteristic
fingerprint based on gingerol derivative analysis for discrimination of ginger
(Zingiber officinale) according to geographical origin using HPLC-DAD combined
with chemometrics. Food Chemistry, 158, 101–111. https://doi.org/10.1016/j.
foodchem.2014.02.086
Zhao, H. Y., Zhang, S. L., & Zhang, Z. W. (2017). Relationship between multi-element
composition in tea leaves and in provenance soils for geographical traceability. Food
Control, 76, 82–87. https://doi.org/10.1016/j.foodcont.2017.01.006
Zhou, J., Cheng, J., He, W., Wang, L. Y., Liu, X., & Lu, W. Y. (2009). Short
communication: Identification of geographical indication tea with Fisher’s
discriminant classification and principal components analysis. Journal of Near
Infrared Spectroscopy, 17, 159–164. https://doi.org/10.1255/jnirs.837