International Journal of Analytical Mass Spectrometry and Chromatography, 2013, 1, 11-21
http://dx.doi.org/10.4236/ijamsc.2013.11003 Published Online September 2013 (http://www.scirp.org/journal/ijamsc)
Tea Identification through Surface-Assisted Laser
Desorption/Ionization Mass Spectrometry
Wen-Tsen Chen, Huan-Tsung Chang*
Department of Chemistry, National Taiwan University, Taipei, Taiwan
Received July 7, 2013; revised August 9, 2013; accepted September 4, 2013
Copyright © 2013 Wen-Tsen Chen, Huan-Tsung Chang. This is an open access article distributed under the Creative Commons At-
tribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is prop-
erly cited.
ABSTRACT
We have applied surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using titanium dioxide
nanoparticles (TiO2 NPs) as the matrix and captopril (CAP) as internal standard for the determination of the concentra-
tions of theanine and four catechins—catechin, (_)-epigallocatechin (EGC), (_)-epicatechin gallate (ECG), and (_)-epi-
gallocatechin gallate (EGCG). Under the optimal conditions (240 nM TiO2 NPs and 10 μM CAP), this SALDI-MS ap-
proach provides linearity of 0.3 – 80 (r = 0.990), 1.2 – 100 (r = 0.987), 4 – 120 (r = 0.995), 6 – 120 (r = 0.983), and 2 –
120 μM (r = 0.991) for theanine, catechin, EGC, ECG, and EGCG, respectively. The limits of detection (LODs; S/N = 3)
for theanine, catechin, EGC, ECG, and EGCG provided by this SALDI-MS approach are 0.1, 0.35, 1.0, 1.45, and 0.5
μM, respectively. This approach provides spot-to-spot and batch-to-batch variations of less than 10% and 13%, respec-
tively, for the analysis of tea samples. With advantages of simplicity, accuracy, precision, and great reproducibility, we
have applied the SALDI-MS approach for the analysis of tea samples, with identified peaks for theanine, catechin, EGC,
ECG, and EGCG. Tea samples from Taiwan and four other areas have various SALDI-MS profiles, showing their po-
tential for differentiation of tea samples from different sources. Our result also shows that tea samples harvested in dif-
ferent seasons and counties in Taiwan provide significantly different MS profiles. The amounts of theanine and EGC in
the Oolong tea from Lishan are much higher than those in the other tea samples.
Keywords: Tea Samples; TiO2 NPs; SALDI-MS; Catechins; Theanine; Captopril
1. Introduction
Matrix-assisted laser desorption/ionization mass spec-
trometry (MALDI-MS) is a powerful tool for biochemi-
cal analysis, in which analytes undergo soft and efficient
desorption/ionization with a minimum degree of frag-
mentation as a result of rapid energy transfer from
UV-absorbing matrixes [1-3]. Although MALDI-MS has
been successfully used for the analyses of variety of
molecules, especially peptides and proteins, it has sig-
nificant limitation on the analysis of small molecules,
due to the interferences of matrix background ions in the
low molecular weight region (<500 Da) [4,5]. Inhomo-
geneous co-crystallization of analytes with traditional
organic matrixes such as 2,5-dihydroxybenzoic acid
(DHB) usually causes high spot-to-spot and sample-to-
sample variations. To overcome problems of “sweet
spots”, surface-assisted laser desorption/ionization mass
spectrometry (SALDI-MS) has been demonstrated [6-8].
Several nanomaterials, including Au [9-11], Ag [12],
carbon nanotubes [13], SiO2 [14], TiO2 [15], HgTe
nanostructures [16-18], and Fe3O4 [19], have been em-
ployed in SALDI-MS. These NPs absorb energy from
laser irradiation and then transfer it to the analytes to
induce desorption and ionization.
Tea is one of the most popular beverages in the world,
which contains great amounts of flavanols, flavonoids,
polyphenols, and catechins [20,21]. The major tea cate-
chins known to possess biological (antioxidant) activity
are (+)-catechin, (−)-epicatechin (EC), (−)-epigallocate-
chin (EGC), (−)-epigallocatechin gallate (EGCG), and (−)-
epicatechin gallate (ECG). In addition to catechins, tea
contains great amounts of amino acids, caffeine, and
ascorbic acid. Their amounts usually vary depending on
species, season, climate, horticultural conditions, and the
degree of fermentation during the manufacturing process
[22]. Several analytical methods have been applied to
determine important tea components, including nuclear
magnetic resonance (NMR) [23], high performance thin
*Corresponding author.
C
opyright © 2013 SciRes. IJAMSC

W.-T. CHEN, H.-T. CHANG 12
layer chromatography (HPTLC) [24], capillary electro-
phoresis (CE) [25], gas chromatography (GC) [26], high-
performance liquid chromatography (HPLC) [27], liquid
chromatography coupled with mass spectroscopy (LC-
MS) [28], and biosensing [29]. However, these analytical
techniques usually require complicated sample prepara-
tion processes, lengthy analysis, and provide low sample
throughput and/or poor sensitivity. In a previous study,
we successfully applied SALDI-MS using TiO2 NPs as
selective probes and matrices to determine the concentra-
tions of several catechins in tea samples, with limits of
detection (LOD) at the picomole level [30].
In this study, we applied this SALDI-MS technique
using TiO2 NPs as matrices for tea identification. In ad-
dition to MS profiles, the mass signals of several identi-
fied analytes were used to improve the identification. In
order to provide better quantitation, captopril (CAP) was
used as an internal standard. We investigated the effects
of the concentration of the TiO2 NPs and CAP in deter-
mining the sensitivity for the analysis of various tea
samples. The MS profiles for catechins show that our
SALDI-MS approach holds great potential for the identi-
fication of various tea samples, with advantages of sim-
plicity, rapidness, and reproducibility.
2. Experimental
2.1. Chemicals
Titanium(IV) isopropoxide (97%), CAP (≥98%), (+)-
catechin hydrate (≥98%), EGC (≥95%) from green tea,
ECG (≥98%), and EGCG (≥98%) from green tea were
obtained from Sigma Aldrich (St. Louis, MO, USA).
L-Theanine (≥98%) was purchased from Tokyo Chemical
Industry (Tokyo, Japan). Nitric acid (HNO3, 97%) was
purchased from Acros (Geel, Belgium). Formic acid (FA,
99.5%) and acetonitrile (ACN, >99.9%) were obtained
from Aldrich (Milwaukee, WI, USA).
2.2. Preparation of TiO2 NPs
TiO2 NPs were prepared through a sol-gel reaction ac-
cording to a procedure described previously. Titanium
isopropoxide (12.5 mL) was added dropwise to 0.1 M
nitric acid (75 mL) under vigorous stirring at ambient
temperature (25˚C), leading to instantaneous formation
of white precipitate. Immediately after hydrolysis, the
slurry was heated at 80˚C and stirred vigorously for 8 h
to convert the slurry into a sol and then to bring it to a
colloidal solution. The mixture was set aside to cool to
ambient temperature and then filtered through a filter
paper to remove agglomerates. The concentration of the
as-prepared TiO2 NPs was estimated to be 240 μM (2 ×
1017 particles/mL) provided that the titanium isopropox-
ide reacted completely to form TiO2 NPs [30].
2.3. Characterization of TiO2 NPs
A double-beam UV-Vis spectrometer (Cintra 10e; GBC
Scientific Equipment Pty Ltd., Dandenong, Victoria,
Australia) was used to measure the absorbance values of
TiO2 NP solutions in the absence and presence of ana-
lytes under acidic conditions (10 mM nitric acid). The
size of TiO2 NPs and their distribution were further con-
firmed through transmission electron microscopy (TEM)
measurements using an H7100 transmission electron
microscope (Hitachi, Tokyo, Japan) operated at 75 kV.
2.4. Analysis of Tea Samples
Tea samples used in this study include Oolong, Jin Xuan,
and Jadeite, which were collected from Taiwan and other
countries, with a total of 40 samples. The Taiwanese teas
harvested in summer and winter, including Oolong (thir-
teen samples) and Jin Xuan (ten samples), were produced
from counties of Lugu (Nantou), Alishan (Chiayi), Pu-
yuma (Taitung), Pinglin (NewTaipei), Datong (Yilan),
Lishan (Taichung), and Ruisui (Hualien). The contents of
the individual catechins in different types of teas (Oolong,
Jinxuan, Black, and four season powder) from Taiwan
and four other countries—China, Vietnam, Indonesia,
Thailand—were analyzed. All of the tea samples were
provided by Tea Research and Extension Station, Taiwan.
Aliquots (40 mL each) of water at 90˚C was poured
separately onto tea leaves (0.16 g), which were then
stirred for 4 min at 72˚C - 75 ℃. Three batches of tea
solutions were prepared from each tea sample in the
same manner. The first brew of each tea sample was fil-
tered through 0.22-μm membranes and then aliquots (1.0
mL) of the filtrates were diluted 10-fold with ultrapure
water containing 10 μM CAP. Aliquots (10 μL) of the
mixtures were mixed with TiO2 NPs (240 nM, 10 μL) for
10 min, which (0.5 μL) were added separately to the
wells of the MALDI plate. After being dried at ambient
temperature for 40 min, the samples were subjected to
SALDI-MS analyses. Triplicate SALDI-MS analyses
were conducted for each tea brew.
2.5. SALDI-TOF MS
Mass spectrometry experiments were performed in the
reflection negative-ion mode using a Microflex MALDI-
TOF mass spectrometer (Bruker Daltonics, Bremen,
Germany), without any instrumental modification. The
samples were irradiated with a nitrogen laser (output at
337 nm) at 10 Hz. Ions produced by laser desorption
were stabilized energetically during a delayed extraction
period of 200 ns and then accelerated through the time of
flight in the reflection mode before entering the mass
analyzer. The applied acceleration voltage was −20 kV
for the negative-ion mode. To obtain good resolution and
high signal-to-noise (S/N) ratios, the laser fluence was

W.-T. CHEN, H.-T. CHANG 13
set at 105 μJ (slightly higher than the threshold) and each
mass spectrum was generated by averaging over 150
laser pulses.
2.6. Electrospray Ionization Mass Spectrometry
(ESI-MS)
A Bruker micrOTOF-QⅡ mass spectrometer (Bruker
Q-TOF system, hybrid quadrupole-time of flight mass
spectrometry) was operated in the negative mode with a
capillary voltage of 3.5 kV; the dry gas flow rate was
controlled at 4.5 L/min; the nebulizer was controlled at
5.8 psi, and dry temperature was set to 180˚C. Full scan
MS spectra were recorded in the m/z range 150 - 500
with 20 acquisitions per spectrum. To obtain stable
electrospray signals, 50% ACN containing 0.1% FA was
added to each of the injected solutions before ESI-MS
measurement. The tea samples, which were diluted
10-fold (0.1x) of the original tea solutions, were infused
directly at 3 μL/min into the mass spectrometer.
3. Results and Discussion
3.1. TiO2 NPs as Assisted Matrices and Captopril
(CAP) as Internal Standard in SALDI-MS
TiO2 NPs were characterized by the UV-vis absorption
and TEM measurements, showing a characteristic ab-
sorption band at 362 nm and an average diameter of 5 ±
1 nm (100 counts). In the presence of catechins, the color
of TiO2 NP solutions became yellow, with an absorption
band at the wavelength around 400 nm as a result of the
interactions of TiO2 with the enediol compounds [31].
Based on our previous study [30], we found that the op-
timum concentration of TiO2 NPs for the SALDI-MS of
tea identification was 240 nM; low background noise
was generated and great sensitivity for catechins in tea
samples was obtained. Upon increasing the concentration
of TiO2 NPs up to 240 nM, the intensities of the MS sig-
nals of the catechins increased, mainly because of in-
creased capture capability and energy absorption. Loss of
mass-resolution and stronger background signals became
problematic when the concentration of TiO2 NPs was
greater than 240 nM.
Figure 1 displays the chemical structures and molecu-
lar weights of the internal standard and the five major
catechins identified in the tea samples in this study. Fig-
ure 2 displays the mass spectra of the TiO2 NPs (240 nM)
and their mixture with 0.1x tea samples through SALDI-
MS analysis. The MS background in the TiO2 NPs was
quite low, with major background peaks at m/z 255 and
283 that are assigned for [Ti4O3OH – H]- and
[TiC12H28O4 – H]-, respectively. From the MS spectrum
of the representative tea sample, we assign the signals at
m/z 173, 289, 305, 441, and 457 to [theanine – H]-,
[catechin – H]-, [EGC – H]-, [ECG – H]-, and [EGCG –
H]- species, respectively. Only theses analytes are identi-
fied, mainly because of their great amounts in the tea
sample. In addition, only analytes containing enediol
groups can have strong interactions with TiO2 NPs [31].
In other words, they can be trapped effectively, leading
to greater concentration effects. The energy absorbed by
TiO2 NPs can transfer to the analytes effectively when
they are on the surfaces, leading to efficient ionization
[10].
To improve the accuracy and reproducibility for the
determination of the concentrations of catechins in dif-
ferent tea samples through the SALDI-MS approach,
CAP, a drug in modern cardiovascular medicine, was
used as an internal standard. Figure 3 displays the mass
spectra of tea samples in the presence of 5, 10, and 100
μM CAP, respectively. These results reveal that the in-
tensities of the signals of the [EGC – H]- species re-
mained almost constant as the CAP concentration was
increased up to 10 M. In this range, the MS signals of
CAP increased. For example, the intensities of the MS
signals at m/z 216 and 305 for the [CAP – H]- and [EGC
– H]- species were 140 and 480 a. u., respectively, at 5
M CAP, while at 10 μM CAP they were 210 and 460 a.
u., respectively. Further increasing the concentrations of
CAP caused decreases in the intensity of the MS signals
of the [EGC – H]-, mainly because of analyte induced
suppression effect [32]. The intensities of the signals of
the [EGC – H]- species decreased significantly when the
CAP concentration was further increased to 100 μM. The
optimal CAP concentration of 10 μM was selected for
quantitation and reproducibility.
3.2. Sensitivity and Linearity
Under the optimal conditions (240 nM TiO2 NPs and 10
μM CAP), this SALDI-MS approach provided linear
ranges of 0.3 – 80 (r = 0.990), 1.2 – 100 (r = 0.987), 4 –
120 (r = 0.995), 6 – 120 (r = 0.983), and 2 – 120 M (r =
0.991) for theanine, catechin, EGC, ECG, and EGCG,
respectively, based on their MS intensities at m/z 173,
289, 305, 441, and 457, respectively. The limits of
detection (LODs; S/N = 3) for theanine, catechin, EGC,
ECG, and EGCG provided by this SALDI-MS approach
were 0.1, 0.35, 1.0, 1.45, and 0.5 M, respectively,
which were 50, 175, 500, 725, 250 femtomole, respec-
tively. This SALDI-MS approach provided LODs for the
analytes lower than their corresponding concentrations (>
5 mM) in tea samples [20], showing its great potential for
the analysis of tea samples. By using CAP as an internal
standard, this SALDI-MS provided spot-to-spot and
batch-to-batch variations of less than 10% and 13%,
respectively, for the analytes at 10 μM. Minimum pro-
blems associated with sweet spots when using inorganic
matrices have been reported [33,34].

W.-T. CHEN, H.-T. CHANG 14
Figure 1. Chemical structures and m/z values of CAP (internal standard), theanine, and four catechins in the tea samples
identified in this study.
Figure 2. Mass spectra of TiO2 NPs in the (a) absence and (b) presence of Oolong tea solutions (PJK 403) recorded through
SALDI-MS. The tea solution was then diluted 10-fold (0.1x) with water. TiO2 NPs (240 nM) were prepared in 10 mM nitric
acid solution. Equal volume of the dilute tea solution and TiO2 NP solution were mixed, which was then equilibrated for 30
min prior to conducting SALDI-MS analysis. The signals at m/z 255 and 283 were assigned for [Ti4O3OH – H]– and
[TiC12H28O4 – H]–, respectively. The signals at m/z 173, 289, 305, 441, and 457 represent the species [theanine – H]–, [catechin
– H]–, [EGC – H]–, [ECG – H]–, and [EGCG – H]–, respectively. SALDI-MS was performed in a reflection negative-ion mode.
A total of 150 pulsed laser shots were applied under a laser.

W.-T. CHEN, H.-T. CHANG 15
Figure 3. Mass spectra of Oolong tea solutions (PJK 403) in the presence of CAP recorded through SALDI-MS using TiO2
NPs. Concentrations of CAP: (a) 5 uM, (b) 10 uM, and (c) 100 uM. Insets to (a)–(c): mass spectra in the m/z range from 420 to
480 Da. Other conditions are the same as those described in Figure 2.
3.3. Identification of Taiwanese and Foreign Tea
Samples
Having advantages of great sensitivity, reproducibility,
and simplicity, this SALDI-MS approach was applied to
analyze Taiwanese and foreign tea samples. Figure 4
displays four representative MS spectra of the Oolong
and Jin Xuan tea samples. Tables 1 and 2 reveal that the
mass signal ratios of the identified catechins relative to
CAP (internal standard) in Oolong and Jin Xuan tea
samples varied with season and sources. Some of their
ratios (contents) are significantly different from season to
season as marked underlines. We note that Taiwanese
teas harvested during the winter season are considered to
be superior to those in the summer [35]. For the Oolong
teas harvested in summer and winter, their catechins
contents were slightly different, besides those from
Pinglin (PJK 550, PJK 2503) and Lishan (PJK 1069, PJK
2589). The relative theanine/CAP and EGC/CAP MS
signal ratios in the tea samples harvested in the summer
and winter (Table 1) were separately 4.9 and 10.0 for
PJK 550, 1.5 and 3.7 for PJK 2503, 12.0 and 7.4 for PJK
1069, and 8.1 and 3.9 for PJK 2589. We note that their
contents in Oolong teas relative to green teas are much
lower [22]. On the contrary, their contents were quite
divergent in Jin Xuan teas harvested in the summer and
winter seasons (Table 2). For the samples from three
counties (Mingjian, Dongshan, Puyuma), the contents of
the catechins in the tea harvested in winter were two to
five folds higher than that in the summer. For example,
the relative theanine/CAP and EGC/CAP MS signal
ratios from Puyuma were separately 4.9 and 13.5 for PJK
448 (winter harvest), and 2.9 and 3.1 for PJK 2614
(summer harvest). Our results reveal that the signals of
the five components in most of the tea samples decreased
in the order of: EGC > theanine > catechin > EGCG >
ECG. The RSD values of MS signals for these five
components in the tested tea samples from three replicate
intra-day and inter-day measurements were 9.2% and
10.5%, respectively. This approach provides advantages
of simplicity, accuracy, precision, and great repro-
ducibility for the detection of catechins in the tea
samples.
To further explore the features of this SALDI-MS ap-
proach, we investigated its ability to analyze the relative
contents of the catechins in Oolong, Jin Xuan, and Jade-
ite, from four other countries, including China, Vietnam,
Indonesia, and Thailand. Figure 5 displays four repre-
sentative MS spectra. The relative contents of the identi-
fied catechins and theanine in the foreign teas were ob-
viously higher than those in Taiwanese teas, especially
that for theanine; the highest relative signal ratios of
theanine/CAP were up to 22 for TAB 024 (Oolong tea of

W.-T. CHEN, H.-T. CHANG 16
Figure 4. Mass spectra of four tea solutions in the presence of CAP (10 μM) recorded through SALDI-MS using TiO2 NPs.
Tea solutions: (a) PJK 550, (b) PJK 2503, (c) PJK 1069, and (d) PJK 2589. Other conditions were the same as those described
in Figure 2.
Table 1. Contents of theanine and catechins in winter and summer Oolong teas from seven counties in Taiwan through
SALDI-MS.
Tea Samples Tea compositions
Theanine Catechin EGC ECG EGCG
Origin No.
I173/I216c I289/I216 I305/I216 I441/I216 I457/I216
PJK 191a 3.08 ~ 4.21 0.42 ~ 0.55 2.26 ~ 3.26 0.02 ~ 0.03 0.08 ~ 0.12
Lugu, Nantou
PJK 2135b 2.96 ~ 3.88 0.38 ~ 0.48 2.06 ~ 2.93 0.02 ~ 0.03 0.08 ~ 0.10
PJK 403a 2.42 ~ 2.50 0.46 ~ 0.52 1.76 ~ 2.25 0.11 ~ 0.15 0.46 ~ 0.55
Alishan, Chiayi
PJK 2194b 2.12 ~ 2.40 0.41 ~ 0.48 1.56 ~ 2.11 0.09 ~ 0.13 0.38 ~ 0.42
PJK 454a 2.16 ~ 2.71 0.71 ~ 0.78 5.24 ~ 5.92 0.06 ~ 0.07 0.12 ~ 0.18
Puyuma, Taitung
PJK 2581b 1.16 ~ 1.31 0.87 ~ 1.28 5.54 ~ 6.13 0.05 ~ 0.07 0.16 ~ 0.20
PJK 550a 3.95 ~ 4.90 0.81 ~ 0.90 8.87 ~ 10.0 0.11 ~ 0.17 0.19 ~ 0.28
Pinglin, NewTaipei
PJK 2503b 1.15 ~ 1.50 0.31 ~ 0.52 2.97 ~ 3.70 0.09 ~ 0.16 0.13 ~ 0.22
PJK 1018a 2.02 ~ 3.03 0.89 ~ 1.25 4.00 ~ 5.31 0.04 ~ 0.05 0.08 ~ 0.14
Datong, Yilan
PJK 1409b 1.02 ~ 1.22 0.95 ~ 1.35 4.21 ~ 4.71 0.03 ~ 0.04 0.02 ~ 0.03
PJK 1069a 8.46 ~ 12.0 0.86 ~ 0.98 6.80 ~ 7.42 0.06 ~ 0.07 0.18 ~ 0.28
Lishan, Taichung
PJK 2589b 6.95 ~ 8.12 0.41 ~ 0.48 3.22 ~ 3.90 0.04 ~ 0.06 0.54 ~ 0.78
Ruisui, Hualien PJK 948a 2.85 ~ 4.04 0.30 ~ 0.42 3.25 ~ 4.12 0.02 ~ 0.03 0.02 ~ 0.03
a. winter harvests (November, 2011); b. summer harvests (April, 2011); c. Signal ratio was calculated from the average values of nine measurements (three
replicate intra-day and inter-day measurements) ± standard deviation (SD). Internal standard (m/z 216): 10 uM CAP.

W.-T. CHEN, H.-T. CHANG 17
Table 2. Contents of theanine and catechins in winter and summer Jin Xuan teas from seven counties in Taiwan through
SALDI-MS.
Tea Samples Tea composition
Theanine Catechin EGC ECG EGCG
Origin No.
I173/I216c I289/I216 I305/I216 I441/I216 I457/I216
PJK 143a 0.34 ~ 0.41 0.43 ~ 0.51 2.86 ~ 3.39 0.03 ~ 0.04 0.20 ~ 0.25
Mingjian, Nantou PJK 2239b 0.25 ~ 0.28 0.12 ~ 0.18 0.89 ~ 0.95 0.03 ~ 0.03 0.05 ~ 0. 0 6
PJK 909a 3.79 ~ 4.62 0.68 ~ 0.74 4.83 ~ 5.50 0.01 ~ 0.03 0.20 ~ 0.30
Dongshan, Yilan PJK 2328b 0.93 ~ 1.05 0.38 ~ 0.47 3.13 ~ 3.62 0.01 ~ 0.02 0.40 ~ 0.54
PJK 448a 4.48 ~ 4.93 2.13 ~ 2.60 12.19 ~ 13.48 0.04 ~ 0.05 0.60 ~ 0.67
Puyuma, Taitung PJK 2614b 2.21 ~ 2.87 0.23 ~ 0.50 2.39 ~ 3.13 0.04 ~ 0.05 0.52 ~ 0. 9 4
Zhushan, Nantou PJK 0075a 7.88 ~ 8.52 0.78 ~ 0.85 4.06 ~ 4.85 0.03 ~ 0.04 0.32 ~ 0.39
Pinglin, NewTaipei PJK 570a 0.88 ~ 0.97 0.40 ~ 0.48 5.95 ~ 6.73 0.02 ~ 0.03 0.18 ~ 0.26
Ruisui, Hualien PJK 1036a 3.78 ~ 4.54 0.31 ~ 0.40 2.79 ~ 3.40 0.02 ~ 0.03 0.17 ~ 0.22
Alishan, Chiayi PJK 1136a 8.93 ~ 9.82 0.45 ~ 0.52 2.09 ~ 2.56 0.02 ~ 0.03 0.18 ~ 0.26
a. winter harvests (November, 2011); b. summer harvests (April, 2011); c. Signal ratio was calculated from the average values of nine measurements (three
replicate intra-day and inter-day measurements) ± standard deviation (SD). Internal standard (m/z 216): 10 uM CAP.
Figure 5. Mass spectra of four tea solutions in the presence of CAP (10 uM) recorded through SALDI-MS using TiO2 NPs.
Tea solutions: (a) TAB 024, (b) TJK 113, (c) TJK 104, and (d) TAB 027. Other conditions were the same as those described in
Figure 2.
summer harvest in Indonesia, Table 3) and for TJK 113
(Jin Xuan tea of winter harvest in Sumatra, Indonesia,
Table 4). The EGC contents in the foreign teas were
usually 1.5 to 20 folds higher than those in Taiwanese
teas. The maximum relative signal ratio of EGC/CAP
was about 20 as shown in TJK 104 (Jin Xuan tea in Bao
Loc, Vietnam), while it was about 0.5 in TJK 113 (Jin
Xuan tea in Sumatra, Indonesia). The contents of EGCG
in the foreign teas relative to the Taiwanese teas were
higher (up to 380-folds). The relative signal ratio of

W.-T. CHEN, H.-T. CHANG 18
Table 3. Contents of theanine and catechins in summer Oolong teas, Four season teas, and Jin Xuan teas from three other
countries through SALDI-MS.
Tea Samplesa Tea composition
Theanine Catechin EGC ECG EGCG
Species Origin No.
I173/I216b I289/I216 I305/I216 I441/I216 I457/I216
Yongfu, China TAB 023 3.35 ~ 3.68 1.17 ~ 1.26 8.04 ~ 8.93 0.13 ~ 0.14 0.15 ~ 0.16
Indonesia TAB 024 20.40 ~ 22.37 0.29 ~ 0.33 7.89 ~ 8.75 0.15 ~ 0.16 0.17 ~ 0.19
North Vietnam TAB 025 12.89 ~ 14.14 1.70 ~ 1.88 13.06 ~ 14.77 0.16 ~ 0.18 0.29 ~ 0.31
Oolong
South Vietnam TAB 027 4.21 ~ 4.79 2.42 ~ 2.69 12.25 ~ 13.52 0.63 ~ 0.70 1.64 ~ 1.81
Dalat, Vietnam VAB 13 4.25 ~ 4.67 0.87 ~ 0.96 7.25 ~ 7.99 0.22 ~ 0.24 0.75 ~ 0.82
Four season Bao Loc, Vietnam VAB 22 5.75 ~ 6.29 0.62 ~ 0.68 8.04 ~ 8.83 0.12 ~ 0.13 1.66 ~ 1.81
Bao Loc, Vietnam VAB 21 0.95 ~ 1.05 0.84 ~ 0.91 6.19 ~ 6.83 0.11 ~ 0.12 0.11 ~ 0.12
Jin Xuan North Vietnam TAB 026 0.75 ~ 0.83 0.61 ~ 0.68 8.05 ~ 8.90 0.10 ~ 0.11 0.31 ~ 0.34
a. summer harvests (April, 2011); b. Signal ratio was calculated from the average values of nine measurements (three replicate intra-day and inter-day meas-
urements) ± standard deviation (SD). Internal standard (m/z 216): 10 uM CAP.
Table 4. Contents of theanine and catechins in winter Jadeite tea, Oolong teas, and Jin Xuan teas from three countries
through SALDI-MS.
Tea Samplesa Tea composition
Theanine Catechin EGC ECG EGCG
Species Origin No.
I173/I216b I289/I216 I305/I216 I441/I216 I457/I216
Jadeite Bao Loc, Vietnam TJK 107 0.58~0.61 0.62~0.64 2.14~2.35 0.088~0.095 0.25~0.26
Mae Salong, Thailand TJK 90 3.54~3.89 0.23~0.25 4.05~4.47 0.13~0.14 0.22~0.23
Medan, Indonesia TJK 92 6.52~7.03 0.71~0.80 12.29~13.77 0.22~0.25 0.35~0.37
Dalat, Vietnam TJK 93 1.28~1.41 0.21~0.23 2.57~2.78 0.067~0.073 0.38~0.41
Oolong
Sumatra, Indonesia TJK 103 14.81~16.68 0.36~0.42 9.19~10.56 0.21~0.24 1.13~1.35
Bao Loc, Vietnam TJK 95 1.12~1.24 0.36~0.39 5.79~6.47 0.084~0.099 0.10~0.12
Moc Chau, Vietnam TJK 98 1.10~1.24 0.85~0.93 10.37~11.13 0.064~0.069 0.70~0.77
Bao Loc, Vietnam TJK 104 13.70~14.90 1.34~1.49 18.17~19.89 0.36~0.41 6.78~7.59
Jin Xuan
Sumatra, Indonesia TJK 113 20.03~21.92 0.14~0.15 0.46~0.50 0.11~12 0.14~0.15
a. winter harvests (November, 2011); b. Signal ratio was calculated from the average values of nine measurements (three replicate intra-day and inter-day
measurements) ± standard deviation (SD). Internal standard (m/z 216): 10 uM CAP.
EGCG/CAP was 7.6 for TJK 104 (Jin Xuan tea in Bao
Loc, Vietnam), while the ratios were 0.02 - 0.94 in the
Taiwanese teas. Except for theanine, EGC, and EGCG,
we also compared with the contents of catechin and ECG.
The contents of catechin (at m/z 289) were not signifi-
cantly different among these teas. On the other hand, the
relative contents of ECG (at m/z 441) in the foreign teas
were higher (up to 70-folds) than those in Taiwanese teas.
The highest relative signal ratio (0.7) of ECG/CAP was
found in TAB 027 (Oolong tea in Vietnam), while the
relative ratios in the Taiwanese teas were only 0.01 -
0.17.
To support our SALDI-MS approach for the analysis
of catechins in teas, ESI-MS analysis of the tea samples
was conducted. Figure 6 displays four representative
mass spectra of PJK 550 (Oolong tea in Pinglin), PJK
1069 (Oolong tea in Lishan), PJK 143 (Jin Xuan tea in
Mingjian), and PJK 448 (Jin Xuan tea in Puyuma). We
assign the peaks at m/z 173, 289, 305, 441, and 457 to
[theanine – H]-, [catechin – H]-, [EGC – H]-, [ECG – H]-,
and [EGCG – H]- species, respectively. When compared
to the ESI-MS and SALDI-MS spectra for the tea sam-
ples, we conclude that the two approaches provided
comparable capability for the identification of catechins
and theanine. Relative to ESI-MS approach, the SALDI-
MS approach is advantageous, including simplicity, ra-
pidity (<10 min), and reproducibility (RSD <11%). It
however provided fewer structural information and
slightly lower sensitivity. These results reveal that the
SALDI-MS approach holds great potential for the identi-
fication of the catechins in various tea samples.
4. Conclusion
We have developed a simple, rapid, and reproducible
SALDI-MS approach for the determination of the con-
centrations of theamine and four catechins using TiO2
NPs as matrices and CAP as internal standard. The
SALDI-MS approach was further validated by the analy-
sis of tea samples from Taiwan and four other areas.

W.-T. CHEN, H.-T. CHANG 19
Figure 6. Mass spectra of four tea solutions of the Oolong teas and Jin Xuan harvested in winter recorded through ESI-MS.
Tea solutions: (a) PJK 550, (b) PJK 1069, (c) PJK 143, and (d) PJK 448. Full scan MS spectra were recorded in the m/z range
150 - 500 with 20 acquisitions per spectrum.
Each sample has its unique SALDI-MS profile, revealing
that the source, harvest region and seasons affect the con-
tents of the analytes. The amounts of theamine in the Jin
Xuan tea samples from Alishan and Zhushan are much
higher than those from other counties (Table 2). Our
rapid and simple SALDI-MS approach reveals that the
amounts of ECG and EGCG in Taiwanese Oolong tea
samples are lower than those from other countries. Our
preliminary results suggest that our SALDI-MS profiles
shall be useful for the identification of tea samples.
5. Acknowledgements
This study was supported by the National Science Coun-
cil of Taiwan under contracts NSC 101-2113-M-002-
002-MY3 and NSC 99-2923-M-002-004-MY3. We thank
the Tea Research and Extension Station, Taiwan, for
providing the tea samples.
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