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Food Chemistry 147 (2014) 262268

Contents lists available at ScienceDirect

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

Analytical Methods

Rapid tea catechins and caffeine determination by HPLC using


microwave-assisted extraction and silica monolithic column
A.A. Rahim a,, S. Nofrizal a, Bahruddin Saad a,b
a
b

School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
Doping Control Center, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia

a r t i c l e

i n f o

Article history:
Received 11 October 2011
Received in revised form 11 September 2013
Accepted 25 September 2013
Available online 3 October 2013
Keywords:
Catechin
Monolithic column
High performance liquid chromatography
Green tea
Oolong tea
Black tea

a b s t r a c t
A rapid reversed-phase high performance liquid chromatographic method using a monolithic column for
the determination of eight catechin monomers and caffeine was developed. Using a mobile phase of
water:acetonitrile:methanol (83:6:11) at a ow rate of 1.4 mL min1, the catechins and caffeine were isocratically separated in about 7 min. The limits of detection and quantication were in the range of 0.11
0.29 and 0.330.87 mg L1, respectively. Satisfactory recoveries were obtained (94.2105.2 1.8%) for all
samples when spiked at three concentrations (5, 40 and 70 mg L1). In combination with microwaveassisted extraction (MAE), the method was applied to the determination of the catechins and caffeine
in eleven tea samples (6 green, 3 black and 2 oolong teas). Relatively high levels of caffeine were found
in black tea, but higher levels of the catechins, especially epigallocatechin gallate (EGCG) were found in
green teas.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Tea is one of the most frequently consumed beverages in the
world, dating back to more than 5000 years ago. Numerous studies
have recorded the benecial effects of tea, e.g., anti-oxidant (Vinson & Dabbagh, 1998; Yen & Chen, 1995), anti-carcinoma (Sadzuka,
Sugiyama, & Sonobe, 2000) and arteriosclerosis prevention (Kritz &
Sinzinger, 1997). The major nutraceuticals in teas are the catechins. There is already growing evidence that tea polyphenols reduce the risk of heart diseases and cancer in humans (Crespy &
Williamson, 2004). In some studies, tea has been associated with
antiallergic action (Sano, Suzuki, Miyase, Yoshino, & MaedaYamamoto, 1999) and antimicrobial properties (Greenwalt,
Ledford, & Steinkraus, 1998; Vaquero, Alberto & Nandra, 2007).
Further studies have demonstrated that the co-administration of
drugs with catechins (C), epicatechin (EC) and epigallocatechin gallate (EGCG) inhibits glucuronidation and sulfation of orally administered drugs thereby increasing the bioavailability of such drugs
(Prasain & Barnes, 2007). Moreover some epidemiological studies
have linked the consumption of tea with a lower risk of several
types of cancer including those of the stomach, oral cavity, oesophagus and lungs (Hakim, Harris, Chow, Dean, Brown & Ali, 2004).
Therefore, tea appears to be an effective chemopreventive agent

Corresponding author. Tel.: +60 4 653388; fax: +60 4 6574854.


E-mail address: adah@usm.my (A.A. Rahim).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.09.131

for toxic chemicals and carcinogens (Karori, Wachira, Wanyoko,


& Ngure, 2010).
The weight percentage of the soluble ingredients account for as
much as 30% of tea, which differs according to variety, production
area, climate, and processing conditions (Bronner & Beecher, 1998;
Han, Tian, & Chen, 1997; Lu, Chu, Yan, & Chen, 2009). The content
of soluble ingredients in green tea is generally greater than those in
black tea as the latter is completely fermented. Polyphenols constitute the major portion of the soluble ingredients and are also the
essential components of tea which have physiological functions.
Catechins are the primary polyphenols in the tea, and accounts
for 7580% of the soluble ingredients (Lu et al., 2009).
Caffeine is the major alkaloid of tea, present in the range of
3.04.0% (Fernandez, Martin, Gonzalez, & Pablos, 2000; Naik &
Nagalakshmi, 1997; Zhang, Lin, He, & Petteruti, 2002). In humans,
caffeine stimulates the heart (Ammon, 1991; Ashihara, Sano, &
Crozier, 2008), central nervous system (Davis, Zhao, Stock, Mehl,
Buggy & Hand, 2003; Nehlig, Daval, & Debry, 1992), and the respiratory system (Doherty & Smith, 2005; Richmond, 1949). It is a
diuretic and has the effect of delaying fatigue (Grandhi, Donnelly,
& Rogers, 2007; Haskell, 1926). The structures of the various
catechin monomers and caffeine are shown in Table 1.
High performance liquid chromatography (HPLC) is by far the
most popular method for the analysis of tea catechins, gallic acid,
purine alkaloids, theanine, etc. (Peng, Song, Shi, Li, & Ye, 2008). In
these methods, the stationary phase used is based on particulate
packing materials (mainly C18 with 5 lm particle size). Particulate

A.A. Rahim et al. / Food Chemistry 147 (2014) 262268


Table 1
Structure of catechin monomers and caffeine.
R, S conformation

R, R conformation

OH

OH
OH

OH
HO

O
A

HO

O
A

C
OH

OH

OH

OH

Epicatechin (EC)

Catechin (C)

OH

OH
OH
HO
A

OH

HO

OH

O
A

OH

OH

C
OH

OH

OH

Gallocatechin (GC)

Epigallocatechin (EGC)
OH

OH
OH
HO
A

O
C

HO
A

O
C

O
OH

OH

B
O

OH

OH

OH

OH
OH

Catechin gallate
(CG)

OH

Epicatechin gallate
(ECG)

OH

OH
OH

OH
HO

O
A

OH

C
O

Gallocatechin gallate
(GCG)

O
C

OH

OH
D
OH

OH

OH

OH
D
OH

N
O

achieved compared to the HPLC method (Arce, Rios, & Valcarcel,


1998). Ultra high performance liquid chromatography (UPLC) represents another interesting development in separation science
(Klejdus, Vacek, Lojkov, Benesova, & Kuban, 2008). The higher
separation efciency made possible by using sub-2 lm particle
stationary phase allows faster separation without compromising
the resolution (Guillarme, Nguyen, Rudaz, & Veuthey, 2007). UPLC
methods for the determination of avonoids and phenolic acids
have been developed (Gruz, Novak, & Strnad, 2008). The UPLC separation of 29 phenolic compounds (including eight catechins) was
feasible in about 20 min (Novkov, Spcil, Seifrtov, Opletal, & Solich, 2010). However, the UPLC unit is costly and requires higher
maintenance costs.
Generally, before analytes are ready for the analytical determination, they need to be isolated from the sample. Liquidliquid
extraction, reux and ultrasonic treatment for the extraction of
catechins and caffeine in tea samples were reported (Buqing, Huiling, Qingling, Buchang, & Shengfang, 1996; Dawidowicz & Wianowska, 2005). While effective, these conventional sample
preparation techniques are laborious, time-consuming, and require
large amounts of solvents. Microwave-assisted extraction (MAE)
has received considerable attention as an alternative extraction
technique (Spigno & De Faveri, 2009). The technique is viewed as
green as the extraction is carried out in vessels in a closed environment, and has been demonstrated to be time- and energy-saving and highly efcient. It has been widely used for the extraction
of analytes from plants and herbs (Li, Huang, Tang, & Deng, 2010).
In the present studies, a new HPLC method using a monolithic column was developed for the simultaneous determination of eight
catechins and caffeine in tea. In conjunction with the MAE, the
method was applied to the determination of catechins and caffeine
in several green, black and oolong teas. The requirement of rapid
methods for determination of catechins in the standardization of
nutraceuticals, clinical, industrial processing of tea beverages and
evaluation of stability of catechin in bakery products are some
other justications for the development of a rapid analytical method (Nishitani & Sagesaka, 2004).

OH

Epigallocatechin gallate
(EGCG)

CH3
N
N

O
H3C

HO

B
O

O
OH

OH

263

N
CH3

Caffeine

packing materials, however, are beset by problems of backpressures when higher ow rates are attempted. Separation times of
20 min or more is often required. Monolithic columns represent
an innovative type of column for rapid chromatographic analysis.
In contrast to the conventional HPLC columns, monolith columns
are formed from a single piece of porous silica gel, thus giving them
greater porosity and permeability, allowing chromatographic analyses to be performed in a fraction of the time previously required
(Neue et al., 2007).
A comparative study between the HPLC and capillary electrophoresis (CE) technique for the separation of catechins in tea has
been reported (Bonoli, Pelillo, Toschi, & Lercker, 2003; Lee & Ong,
2000). The CE is in certain ways advantageous due to the shorter
analysis time and reduced consumption of solvents and sample;
but is nevertheless less sensitive due to the short sample path
length of the CE capillary. In addition, the consistency and reproducibility of the CE method were much more difcult to be

2. Materials and methods


2.1. Materials
Caffeine, methanol and acetonitrile of HPLC grade were purchased from Merck (Darmstadt, Germany). () catechin (C), ()
epicathecin (EC), () epigallocatechin gallate (EGCG), () gallocatechin gallate (GCG), () epicathechin gallate (ECG), ()- catechin gallate (CG), ()-gallocatechin (GC) and ()-epigallo
catechin (EGC) standards were purchased from Sigma Aldrich
(Steinheim, Germany). Tea samples were purchased from supermarkets in Penang, Malaysia and were produced from Malaysia,
United Kingdom, Japan, Taiwan, Indonesia and China.
2.2. Instrumentation and chromatographic conditions
The HPLC system consisted of two LC-10 AD VP pumps and
SPD-10Avp UV detector (Shimadzu, Japan). The mobile phase
composition that comprises of water:acetonitrile:methanol with
compositions of 85:6:9, 84:6:10, 83:6:11 were studied at different
ow rates (1.0, 1.2 and 1.4 mL min1). Monolithic Rp-18 e 100
4.6 mm (Merck KGaA, Germany) and BDS Hypersil gold C-18
(4.6 mm I.D.  250 mm) columns were used. Signals were monitored at 280 nm. Standard solutions and tea samples were ltered
through a 0.45 lm acrylic polymer lter before being injected into
the HPLC unit. Peak areas versus concentrations were plotted using
linear least-squares analysis. 20 lL of samples were injected.

264

A.A. Rahim et al. / Food Chemistry 147 (2014) 262268

Quantication of the analytes was based on the peak area using the
external calibration method.
2.3. Analytical characteristics
2.3.1. Linearity
Linearity of the curves was established by injecting standard
mixtures of catechins and caffeine (0.1100 mg L1). Linear least
squares regression was used to calculate the coefcient of determination (R2).
2.3.2. Limit of detection (LOD) and limit of quantication (LOQ)
The LOD and LOQ were determined as follows:

LOD 3  SD=slope
LOQ 10  SD=slope
where SD is the standard deviation.
2.3.3. Repeatability and reproducibility studies
The repeatability and reproducibility of the peak areas were
investigated by injecting each of the standard mixtures (5, 40
and 70 mg L1) on the same day (intra-day) and over ve days
(inter-day), respectively.
2.3.4. Recovery studies
Recovery studies were carried out by spiking three concentrations (5, 40 and 70 mg L1) of a mixture of catechins and caffeine
standards to each type of tea samples studied.
2.4. Optimisation of microwave assisted extraction (MAE)
The tea sample (0.5 g) was placed into an extraction tube
(100 mL) and 25 mL of either (i) acetonitrile:water (1:1), (ii) methanol:water (1:1) or (iii) water:acetonitrile:methanol, (83:6:11)) solvent was added. The extraction tube was put into a microwave
unit (Mars 5, CEM Corporation, USA). The power was set at 300 or
600 W. The suspensions were irradiated with microwave as follows:
210 min (heated to about 80 C at 250, 300, 350 or 400 Psi). It was
allowed to cool for about 15 min. The extract was transferred to a
10 mL volumetric ask (10 mL) and made up to the mark using the
appropriate solvent. The mixture was ltered using nylon syringe
lter (0.45 lm, 13 mm diameter) (Gema Medical, Germany).
3. Results and discussion
3.1. HPLC method development
3.1.1. Effect of mobile phase compositions
The effect of mobile phase compositions viz., (i) water:acetonitrile:methanol (85:6:9); (ii) 84:6:10 and (iii) 83:6:11 were studied
at a xed ow rate (1.0 mL min1). When the rst mobile phase
was used, catechin and caffeine were not well resolved. All the catechins and caffeine were separated when the second composition
was used, however the separation time was relatively long (about
14 min). The third mobile phase provided good separation of the
components in about 12 min, and was thus used for the remaining
studies. The order of elution was: GC > EGC > C > caffeine > EGCG > EC > GCG > ECG> and >CG. As compared to the mobile phases mentioned in the works of Wang, Helliwell, and You
(2000), Fernandez et al. (2000) and Nishitami and Sagesaka
(2004), the present study was found to be the most suitable in
the analysis of catechin monomers and caffeine in tea samples.
For example, Wang et al. (2000) used a mobile phase of acetonitrile:water:ortho phosphoric acid (10:89.9:0.1) for the isocratic

separation and determination of catechin monomers and caffeine.


A reasonable separation with distinct multiple peaks was achieved
for gallic acid, GC, caffeine, EGCG, EC, GCG and ECG with this solvent system. However, EGC and C monomers were eluted as a single peak and furthermore required longer analysis time (50 min).
3.1.2. Effect of ow rates
The effect of ow rates (1.0, 1.2 and 1.4 mL min1) on the separation of the analytes were studied. It was noted that retention
time decreased with increase in ow rate and all the peaks were
well resolved. Therefore, 1.4 mL min1 was chosen.
The adopted HPLC conditions were: isocratic elution using mobile phase composition of water:acetonitrile:methanol (83:6:11);
ow rate, 1.4 mL min1; monitored at 280 nm. Under these conditions, all the analytes were separated in less than 7 min. As comparison, the analytes were also separated using a particulate DBS
Hypersil C-18 column (Fig. 1a) with the same HPLC conditions.
The use of this column resulted in good separations, but the separation time was longer (about 30 min). Similar order of elution to
that of the monolithic column was found (Fig. 1b), suggesting similar separation mechanism between the columns.
3.2. Analytical characteristics
The results of developed HPLC method were validated in terms
of linearity, LOD, LOQ, precision and recovery.
3.2.1. Linearity
Linear calibration curves were obtained by plotting the peak
area against the concentration of the respective standards and
were found to be linear over the range 0.180 mg L1 (Table 2).
All the analytes showed good linearity with coefcient of determination (R2) ranging from 0.9989 to 0.9998 for the eight catechins
standards and caffeine.
3.2.2. Limit of detection (LOD) and limit of quantication (LOQ)
A blank sample that was spiked with 1 mg L1 of each standard
was used for the measurement of LOD and LOQ. Calibration curve
was constructed, the standard deviation and slope were used to
calculate the LOD and LOQ. The LOD and LOQ for these catechins
and caffeine (Table 2) were comparable to that reported by Yang,
Ye, Xu, and Jiang (2007) and Wang, Provan, and Helliwell (2003).
3.2.3. Repeatability and reproducibility studies
The repeatability of the peak area was assessed by injecting
mixtures of each standard (n = 6). The relative standard deviation
(RSD) for the peak area was found to range between 0.76% and
2.53% (Table 2). The reproducibility over different days was carried
out by injecting the same standard solution over 5 days. Good
reproducibility of the peak area (RSD 6 2.55%) was found for all
experiments (Table 2).
3.2.4. Recovery studies
Recovery studies were carried out by spiking three concentrations of a mixture of catechins and caffeine standards to each type
of tea samples. Good recoveries of 94.2105.2 1.8% were found
for all the spiked samples (Table 3).
3.3. Comparison with other HPLC methods
Table 4 summarises the main analytical characteristics of some
recent HPLC methods for the determination of catechins and related compounds. The list is by no means exhaustive, but rather
serves to illustrate the important features of the reported HPLC
techniques. As mentioned earlier, all the previous reports used
particle-type stationary phase and gradient elution was preferred

A.A. Rahim et al. / Food Chemistry 147 (2014) 262268

265

Fig. 1. Typical chromatograms for the separation of catechins and caffeine standards using particulate DBS Hypersil gold C-18 (a), monolithic column (b) and green tea (c),
oolong tea (d), black tea (e) samples on monolithic column; isocratic elution with water:acetonitrile:methanol (83:6:11); ow rate,1.4 mL min1. Assignment of peaks: (1)
()-gallocatechin (GC), (2) ()-epigallo catechin (EGC),(3) ()-catechin (C), (4) ()-caffeine, (5) ()-epigallocatechin gallate (EGCG), (6) ()-epicatechin(EC), (7) ()gallocatechin gallate (GCG), (8) ()-epicatechin gallate (ECG), and (9) ()- catechin gallate (CG).

as shorter analysis times and improved resolution can be realised.


The superiority of our proposed method based on monolithic column due to the short separation time (7 min) is evident. Separation
times rivaling to those of UPLC systems can be realised by employing suitable gradient programmes.
3.4. Optimisation of microwave assisted extraction (MAE)
Several parameters viz., type of solvent, pressure, power and
time for the microwave irradiation were studied and optimised
in order to obtain suitable extraction conditions of the interested

tea components. Supporting information is given as a Supplementary le.


3.4.1. Effect of extracting solvents
The effect of (i) acetonitrile:water (1:1), (ii) methanol:water
(1:1) and (iii) water:acetonitrile:methanol (83:6:11) as extracting
solvents were studied. It is clear that the third extracting solvents
(i.e., the HPLC mobile phase) resulted in the highest extraction of
the components of interest. Hu et al. (2009) also obtained high
extraction efciency for catechin monomers and caffeine when
methanol:acetonitrile (1:1) were used. On the other hand, our

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A.A. Rahim et al. / Food Chemistry 147 (2014) 262268

Table 2
Analytical characteristics of the developed HPLC method.
Analyte

R2

Linear range (mg L1)

LOD (mg L1)

LOQ (mg L1)

Intra-day (%RSD) n = 6

Inter-day (%RSD) n = 5
1

Concentration spike, mg L

GC
EGC
Catechin
Caffeine
EGCG
EC
GCG
ECG
CG

0.580
0.580
0.580
0.580
0.580
0.580
0.580
0.580
0.580

0.9997
0.9994
0.9998
0.9991
0.9998
0.9992
0.9997
0.9995
0.9994

0.21
0.24
0.29
0.17
0.22
0.13
0.22
0.27
0.11

0.63
0.72
0.87
0.51
0.66
0.39
0.66
0.81
0.33

40

80

40

80

2.27
2.00
2.26
2.11
1.76
1.92
1.66
1.89
2.05

1.02
2.14
1.59
1.78
1.84
1.61
1.77
0.76
1.00

1.88
1.78
2.53
1.97
2.44
1.89
2.17
2.53
2.35

1.93
1.96
2.39
1.75
1.81
2.17
2.32
1.23
2.49

1.98
2.22
1.78
1.89
0.89
1.95
1.62
1.77
1.09

1.47
2.28
2.21
2.27
1.49
1.88
2.53
2.22
1.98

R2, square of regression coefcient; RSD, %relative standard deviation.

Table 3
Recoveries of catechins and caffeine in green, black and oolong tea samples.
Concentration
spike, mg L1

Type

Analytes (%)
EGC

Catechin

Caffeine

EGCG

GCG

ECG

CG

Green tea (#1)


Black tea (#7)
Oolong tea (#10)

97.4 1.2
98.1 1.8
97.1 2.9

96.6 1.9
94.4 2.9
100.8 2.0

98.8 1.4
94.9 1.4
105.2 2.5

100.2 0.8
96.6 2.3
102.1 1.8

102.5 2.0
99.2 0.8
100.2 0.7

99.9 2.1
95.7 1.7
99.4 1.8

101.5 0.9
96.1 2.4
95.7 2.2

99.4 2.2
100.7 2.0
98.3 1.9

100.4 1.6
102.2 2.7
101.1 0.6

40

Green tea (#1)


Black tea (#7)
Oolong tea (#10)

102.3 2.3
96.7 0.8
95.7 1.9

103.2 1.9
98.1 1.2
98.5 1.1

96.3 2.6
97.2 2.3
100.3 2.1

96.9 1.1
104.2 1.3
101.6 2.9

97.3 2.5
101.5 0.8
105.3 1.8

104.2 0.8
100.9 1.5
97.8 0.8

96.1 1.5
102.3 2.5
99.1 1.7

99.1 1.1
96.4 2.7
102.4 2.1

95.8 2.1
95.4 1.9
98.7 0.7

70

Green tea (#1)


Black tea (#7)
Oolong tea (#10)

103.9 2.1
97.6 1.1
101.5 1.9

97.3 1.6
105.2 0.9
102.9 2.1

100.1 2.7
100.2 2.1
99.7 2.1

96.3 1.9
102.6 0.4
98.6 2.2

95.8 2.0
103.3 2.1
94.5 1.8

99.5 2.2
101.7 1.6
96.8 2.7

94.6 2.8
96.3 0.3
97.9 2.9

94.2 1.1
99.0 0.8
99.2 1.1

95.1 2.0
99.1 2.6
95.7 0.7

GC

EC

# = sample number (Table 5).

Table 4
Some reported HPLC methods for the analysis of tea avonoids and phenolic acids.
No. Analytes

Detector Column

Dimension

Program LOD

LOQ

Separation References
Time
(min)

1.

Catechins

PDA

Wakosil-II 5 C-18

3 mm  150 mm, 5 lm

Gradient 0.51.2 ng

40

2.

Phenolic acid, purine,


theanine
Catechins, purine,
gallic acid
Catechins
Catechins,
theaavines
Catechins, caffeine,
gallic acid
Catechins

UV

RP-Amine C-16

4.6 mm  150 mm, 5 lm

Gradient 0.22.8 ng

0.76.3 ng

45

Nishitani and Sagesaka


(2004)
Peng et al. (2008)

DAD

ODS-100Z C-18

3.9 mm  100 mm, 5 lm

Gradient 0.040.89 ng

11

Hu et al. (2009)

UV
UV

C18 packing
Partispere C-18

3.9 mm  150 mm, 5 lm


4.6 mm  110 mm, 5 lm

Isocratic

Gradient 0.05 lg mL1

40
25

Row and Jin (2006)


Lee and Ong, (2000)

UV

Isocratic

50

Wang et al. (2000)

DAD

Kingsorb/Nucleosil 4.6 mm  150 mm, 5 lm


C-18
C-18 packing
4.6 mm  150 mm, 5 lm

Wang et al. (2003)

DAD

Mightysil Rp-18

4.6 mm  150 mm, 5 lm

20

Yang et al. (2007)

UV

Monolithic C-18

4.6 mm  100 mm

0.643.86
mg L1
2.28.5 mg
L1
0.33
0.87 mg L1

25

Purine alkaloide,
catechins
Catechins and
caffeine

Isocratic 0.19
1.16 mg L1
Gradient 0.842.98
mg L1
Isocratic 0.110.29
mg L1

3.
4.
5.
6.
7.
8
9

Our method

PDA, photo diode array; UV, ultraviolet visible spectrometry; DAD, diode array detector.

results were found to be better than the one reported by Nishitani


and Sagesaka (2004) who studied acetonitrile:water (1:1) as the
extracting solvent.

3.4.2. Effect of microwave parameters


The extraction efciency of catechins and caffeine in tea samples that were extracted using different pressures (250, 300, 350
and 400 Psi) were studied. The compounds were optimally

extracted using 350 or 400 Psi, but the lower pressure was used
for the studies.
The tea samples were extracted at different irradiation power
(300 or 600 W) for 6 min. The best extraction was when 600 W
was used. Varying microwave irradiation times (2, 4, 6, 8 and
10 min) using 600 W was then investigated. It was observed that
the extraction efciency for the catechins and caffeine increased
with irradiation time until 6 min and remained stable after
810 min. This value was lower than that reported by Pan, Niu,

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A.A. Rahim et al. / Food Chemistry 147 (2014) 262268


Table 5
Levels of catechins and caffeine in tea samples analysed.
Concentration (mg g1)

Samples

GC

EGC

Catechin

Caffeine

EGCG

EC

GCG

ECG

CG

GT

1
2
3
4
5
6

4.24 0.66
5.32 0.11
5.04 0.17
3.76 0.13
4.44 0.23
6.24 0.55

17.0 0.23
17.9 0.39
10.9 0.22
18.5 0.85
12.2 0.17
13.8 0.15

2.36 0.02
3.56 0.23
2.08 0.08
1.24 0.11
2.28 0.12
2.24 0.05

35.7 0.77
38.7 0.89
24.9 0.32
20.4 0.43
30.2 0.88
32.6 0.23

33.7 0.65
36.1 0.54
27.1 0.41
24.6 0.52
24.5 0.16
28.8 0.19

8.32 0.92
9.08 0.14
6.72 0.18
5.56 0.09
7.08 0.29
7.69 0.05

2.38 0.89
2.53 0.06
0.84 0.02
0.96 0.11
0.92 0.13
1.64 0.36

9.48 0.95
10.2 0.32
7.26 0.86
5.06 0.34
6.12 0.27
8.26 0.25

0.22 0.03
0.28 0.08
0.31 0.05
0.22 0.03
0.17 0.04
0.23 0.01

OT

7
8

6.08 0.13
7.69 0.15

26.2 0.62
24.0 0.41

0.64 0.09
0.88 0.11

41.0 0.83
43.3 0.74

13.8 0.22
15.7 0.33

11.1 0.30
12.9 0.30

1.56 0.06
1.44 0.09

12.4 0.36
14.3 0.55

0.22 0.09
0.19 0.07

BT

9
10
11

0.39 0.02
3.32 0.29
7.69 0.15

1.14 0.05
0.84 0.06
0.49 0.06

0.46 0.08
0.42 0.08
0.85 0.10

69.2 0.91
61.1 0.98
63.3 0.88

5.7 0.22
4.6 0.04
5.7 0.33

1.14 0.06
1.46 0.14
1.92 0.30

0.44 0.04
2.72 0.74
1.44 0.09

4.08 0.54
5.72 0.17
4.27 0.55

0.24 0.05
0.19 0.07
0.25 0.07

GT = green tea; OT = oolong tea; BT = black tea.

and Liu (2003) and Li et al. (2010), when 15 min and 20 min of irradiation time was used, respectively at 400 W.
The adopted microwave extraction conditions were: pressure,
350 Psi; irradiation power, 600 W; irradiation time, 6 min.

3.5. Analysis of tea samples


In the analysis of tea samples, peak identication was based on
the comparison between the retention times of standard compounds and was conrmed by spiking standards to the samples
(Fig. 1ce). Quantication was based on the external standard
method using calibration curves tted by linear regression analysis. The analysis was performed in triplicate.
The MAE and HPLC method were applied for the determination
of several commercial tea samples. The levels of the catechin
monomers and caffeine found are shown in Table 5.
Green tea (samples 16) is a type of tea obtained exclusively
from the leaves of Camellia sinensis that has undergone minimal
oxidation during its production. It was observed that EGCG was
the major catechin monomer found in green tea (a non fermented
tea) in the range of 24.5236.08 mg g1, followed by EGC, ECG, EC,
GC, GCG, catechin and CG. The identied epicatechins (EGCG, EGC,
ECG and EC) are in cis structure. They can be reversibly converted
to their corresponding epimers that are non-epicatechins (GCG, GC,
CG and C). Thus, the chemical structures of epicatechins and nonepicatechins differ only between the 2R, 3R (2,3-cis, epi-form) and
2S, 3R (2,3-trans, non-epi form) (Table 1). The accumulation of
EGCG in green tea is due to inactivation of the polyphenol oxidase
by drying and steaming the fresh leaves until non oxidation process occurs (McKay & Blumberg, 2002). The results shown in Table 5 are generally in agreement with those previously reported
by Yang et al. (2007) and Wang et al. (2003).
Oolong tea (samples 78) is a semi-fermented tea that is produced when the fresh leaves are subjected to a partial fermentation
stage before drying (McKay & Blumberg, 2002). It can be thought of
as being half-way between green and black tea (Sano et al., 1999;
Wang, Lu, Miao, Xie, & Yang, 2008). The results shown in Table 5
are in good agreement with the nding of Zuo, Cheng and Deng
(2002) and Horzic Komes, Belscak, Ganic, Ivekovic and Karlovic
(2009) where the level of catechin monomers in oolong tea was
higher than black tea but relatively lower compared to green tea.
Oolong tea that is rich in antioxidants known as polyphenols
showed high levels of the EGC, ECG, EGCG and EC (R, R conformation) followed by GC, catechin, GCG and CG (R, S conformation).
Comparable results were also reported by Fernndez-Cceres, Martn, Pablos, and Gonzlez (2001) where the EGCG and EGC were the
major catechins found in semi fermented oolong teas.

Fermented black tea (samples 911) undergoes a post-harvest


fermentation stage before drying and steaming processes (McKay
& Blumberg, 2002). The fermentation of black tea is due to an oxidation process that is catalysed by polyphenol oxidase, and also attained by using microorganisms. The level of catechin monomers
in black tea is lower than green tea and oolong tea. This is due to
the alteration of catechin to form theaavin, thearubigins during
fermentation process (Wang et al., 2000).Naturally, all the fresh
tea leaves contain certain levels of caffeine. The levels of caffeine
vary depending on the variety of tea origin, structure of the tea leaf
and the differences in the tea processing itself (Song, Lin, Qu, &
Huie, 2003).
From Table 5, the level of caffeine in fully fermented black tea
was in the range 61.169.2 mg g1, whereas semi fermented oolong tea and non fermented green tea give values in the range of
41.043.3 and 20.438.7 mg g1, respectively.
4. Conclusion
A rapid HPLC method using a monolithic column was developed, validated and applied to the simultaneous determination of
eight catechin monomers and caffeine in tea samples. The use of
the monolithic column enables these analytes to be separated almost four times faster than that of a conventional particulate column (7 min versus 27 min). The attractiveness of the MAE
technique is further demonstrated where signicant improvements in the extraction efciency of the catechins and caffeine
were obtained. In conjunction with MAE, the HPLC method readily
lends itself as a useful analytical technique for the determination of
catechins and caffeine.
Acknowledgement
The authors gratefully acknowledge the nancial support of this
work by the Short Term Grant of Universiti Sains Malaysia (304/
PKIMIA/6310028).
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.
2013.09.131.
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